Anti-viral guanosine-rich oligonucleotides and method of treating HIV

ABSTRACT

A method and compositions for treating viral infection in vitro and in vivo using a guanosine-rich oligonucleotide. The oligonucleotides have sufficient guanosine to form a guanosine tetrad. Also provided are oligonucleotides of at least two runs of at least two guanosines. Also provided are guanosine-rich oligonucleotides and methods for treating viral infections in humans, and a method for designing guanosine-rich oligonucleotides having anti-viral activity and integrase inhibition activity.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/145,704 filed Oct. 28, 1993, now U.S. Pat. No.5,567,604, which is a continuation-in-part of U.S. patent applicationSer. No. 08/053,027 filed Apr. 23, 1993, now abandoned. This applicationis also a continuation-in-part of allowed U.S. patent application Ser.No. 08/535,168, filed Oct. 23, 1995, now U.S. Pat. No. 6,184,369, whichis the U.S. national stage of PCT/US94/04529 filed Apr. 25, 1994. Thepresent application also claims the benefit of the following 35 U.S.C§111(b) provisional applications: Ser. Nos. 60/001,505, filed Jul. 19,1995, 60/013,688, filed Mar. 19, 1996, 60/014,007, filed Mar. 25, 1996,60/015,714, filed Apr. 17, 1996, and 60/016,271, filed Apr. 23, 1996.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to this invention was supported in part by a grant fromthe United States Government through the National Institute of Healthunder a CRADA agreement. The U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of oligonucleotidechemistry and anti-viral pharmacotherapy. More specifically, the presentinvention relates to novel guanosine-rich oligonucleotides and their useas novel anti-viral agents.

2. Description of the Related Art

General In Vitro Studies

Previously, it was believed that “antisense” oligonucleotides inhibitviruses by interfering with protein translation via an RNA:DNA duplexstructure. More recent research, however, indicates a variety ofpossible mechanisms by which oligo-nucleotides inhibit viral infections.For example, oligodeoxycytidine (poly SdC) inhibits HIV-1. Marshall etal., PNAS (1992) 89:6265-6269, discussed the potential mechanism(competitive inhibition) by which oligodeoxycytidine directly inhibitsviral reverse transcriptase. Poly SdC also inhibited AMV reversetranscriptase and Pol I (Klenow fragment) and polymerase α, β and γ.Previously, Matsukura et al., PNAS (1987) 84:7706-7710, used a similarphosphorothioate derivative of oligo-deoxycytidine to demonstrateinhibition of HIV-1 in culture. Marshall and Caruthers, Science (1993)259:1564-1569, reported the use of diphosphorothioate oligo-nucleotides,e.g., antisense-specific, random nucleotide combinations andoligodeoxycytidine against HIV-1. In all cases, the mechanism of actionwas attributed to a direct inhibition of HIV-1 reverse transcriptase.Other potential mechanisms of anti-viral action of oligonucleotides werepostulated by Boiziau et al., PNAS (1992) 89:768-772, e.g., promotion ofRNAse H activity and inhibition of reverse transcriptase initiating cDNAsynthesis. In addition, Goa et al., Molecular Pharmacology (1992)41:223-229 reported that phosphorothioate oligonucleotides inhibit humanDNA polymerases and RNAse H, and the adsorption or penetration of thevirus into cells. Iyer et al., Nucleic Acids Research (1990)18:2855-2859 reported that if a base was removed from an anti-sensepolynucleotide forming an abasic site, the compound did not lose itsactivity which argues against the need for the formation of an RNA:DNAantisense mediated hybrid for anti-viral activity. Stein et al. havecharacterized the interaction of poly SdC with the V3 loop of HIV-1gp120, and postulated that the specific interaction of poly SdC with theHIV-1 V3 loop may be a mechanism by which an oligonucleotide couldinhibit HIV-1 in vivo.

It is known that synthetic oligonucleotides may be designed which arecapable of binding to duplex DNA to form triplex DNA. See U.S. Pat. No.5,176,996 Hogan & Kessler issued Jan. 5, 1993. That patent discloses amethod for making synthetic guanosine-rich oligonucleotides which aretargeted to specific sequences in duplex DNA and which form collineartriplexes by binding to the major groove of the DNA duplex.

Specific In Vitro Studies/In Vitro HIV Inhibition With T30177

Infection with the human immunodeficiency virus type 1 (HIV-1) and thesubsequent development of acquired immunodeficiency syndrome (AIDS), hasbecome a threat to public health on a global scale. Preventing furtherspread of this disease is a major health priority worldwide. AlthoughHIV-1 was confirmed to be the causative agent of AIDS as early as 1984,few drugs and no vaccines are effective at preventing the ultimate onsetof AIDS in HIV-1 seropositive individuals. This is due, in large part,to the complexity of the causative agent itself, the dynamics of virusproduction and the speed at which drug-resistant mutants can arise. Ho,et al., Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122(1995).

Infection of T-cells by HIV-1 results in the insertion of proviral(double-stranded) DNA into the host cell genome. Goff, S. P., Annu. Rev.Genet. 26:527-544 (1992). The integration process involves both thesequence-specific and sequence independent endonucleolytic and strandtransfer activities of the virally encoded integrase enzyme. Katz, etal., Ann. Rev. Biochem. 63:133-173 (1994); Vink, et a., Trends inGenetics 9:433-438 (1993). Once the proviral state is established, theinfection may manifest itself in several ways including a latentinfection in which viral replication is not measurable until the cellbecomes activated or through a chronic infection in which dividing ornon-dividing cells persistently release virus in the absence of anycytopathic effect. In addition, recent reports on the kinetics of virusproduction (and clearance) indicate a dynamic process in which virtuallya complete replacement of wild-type virus by drug-resistant virus inplasma can occur after only two to four weeks of drug therapy. Ho, etal., Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122 (1995).For this reason it is of utmost importance to develop new anti-HIV-1agents which can complement, by additive or synergistic activity,current therapies.

One relatively new approach used in the development of antiviraltherapeutics for HIV-1 is the use of oligonucleotides designed asantisense agents. Letsinger, et al., Proc. Natl. Acad. Sci. USA86:6553-6556 (1989); Lisziewicz, et al., Proc. Natl. Acad. Sci. USA90:3860-3864 (1993); Milligan, et al., J. Med. Chem. 36:1923-1937(1993). While much effort is being spent on rationally designedoligonucleotides such as antisense agents there have also been recentfindings of multiple alternative mechanisms by which oligonucleotidescan inhibit viral infections. Gao, et al., J.B.C. 264:11521-11526(1989); Marshall, et al., Proc. Natl. Acad. Sci. USA 89:6265-6269(1992); Ojwang, et al., J. AIDS 7:560-570 (1994); Rando, et al, J. Biol.Chem. 270:1754-1760 (1995). For example, Stein et al. (Stein, et al.,Antisense Research and Development 3:19-31 (1993)) have characterizedthe interaction of oligodeoxycytidine, containing a phosphorothioate(PT) backbone (poly (SdC)) with the v3 loop of HIV-1 gp 120. It wasdetermined that poly (SdC)₂₈ specifically interacted with the positivelycharged V3 loop with a Kd of approximately 5×10⁻⁷M. Stein et al.(Antisense Research and Development 3:19-31 (1993)) then postulated thatthe interaction of poly (SdC) with the HIV-1 v3 loop may be a mechanismby which poly (SdC) could inhibit HIV-1 in vivo. More recently, Wyattet. al. (Wyatt, et al., Proc. Natl. Acad. Sci. USA 91:1356-1360 (1994))have described the interaction of a short G-rich oligonucleotide,synthesized with a total PT backbone, which also interacts with the v3loop of HIV-1 gp 120. In addition, we have previously reported thatoligonucleotides containing only deoxyguanosine (G) and thymidine (T),synthesized with natural phosphodiester (PD) internucleoside linkages,were capable of inhibiting HIV-1 in culture. Ojwang, et al., J. AIDS7:560-570 (1994). The most efficacious member of he G22 this dG-richclass of oligonucleotides, I100-15, was found capable of folding uponitself to form a structure stabilized by the formation of two stackedguanosine-tetrads which yielded a guanosine-octet. Rando, et al, J.Biol. Chem. 270:1754-1760 (1995). Furthermore, it was observed that thepositions of the guanosine bases in the I100-15 sequence, found in boththe tetrads and connecting loops in that structure, were extremelyimportant to the overall anti-HIV-1 activity of the oligonucleotide.Rando, et al, J. Biol. Chem. 270:1754-1760 (1995).

Site of Activity Studies-Viral Integrase Inhibition

Two events which are characteristic of the life cycle of retrovirusescan be utilized for therapeutic intervention. One is reversetranscription, whereby the single-stranded RNA genome of the retrovirusis reverse transcribed into singled-stranded cDNA and then copied intodouble-stranded DNA. The next event is integration, whereby thedouble-stranded viral DNA generated by reverse transcriptase is insertedinto a chromosome of the host cell, establishing the proviral state.Integration is catalyzed by the retroviral enzyme integrase which isencoded at the 3′-end of the pol gene. Varmus, et al. Mobile DNA, pp.53-108, Am. Soc. Microbiol, Washington, D.C. (1989). Integrase firstcatalyzes the excision of the last two nucleotides from each 3′-end ofthe linear viral DNA, leaving the terminal conserved dinucleotideCA-3′-OH at these recessed 3′ ends (FIG. 23A). This activity is referredto as the 3′-processing or dinucleotide cleavage. After transport to thenucleus as a nucleoprotein complex, Varmus, et al. Mobile DNA, pp.53-108, Am. Soc. Microbiol, Washington, D.C. (1989), integrase catalyzesa concerted DNA strand transfer reaction by nucleophilic attack of thetwo viral ends onto a host chromosome. This reaction generates arecombination intermediate resembling an X structure, analogous to aHolliday junction intermediate. [For recent reviews see Katz and Skalka,Katz, et al., Ann. Rev. Biochem. 63, 133-173 (1994), and Vink andPlasterk, Vink, et al., Trends Genet. 9, 433-437 (1993)]. Mutationanalyses of the viral integrase gene demonstrate that integration isrequired for effective retroviral replication and that it is alegitimate target for the design of antiretroviral drugs (Engleman, etal., J. Virol. 69, 2729-2736 (1995); Englund, et al, J. Virol. 69,3216-3218 (1995)).

It is known that AZT nucleotides can inhibit HIV-1 integrase, Mazumder,et al., Proc. Natl. Acad. Sci. 91, 5771-5775 (1994), and thatsubstitution or unsaturation at the 3′-position of the deoxyriboseconfers potency against HIV-1 integrase. These results suggested thatthe enzyme's nucleotide binding site could serve as a potential drugtarget. It has been shown that the potential stacking interactionsgained from the heterocyclic rings can further enhance potency againstHIV-1 integrase.

Recently, oligonucleotides composed of deoxyguanosine and thymidine havebeen reported to inhibit HIV-1 replication. Rando, et al., J. Biol.Chem. 270, 1754-1760 (1995); Wyatt, et al., Proc. Natl. Acad. Sci.U.S.A. 91, 1356-1360 (1994). Oligonucleotides forming intramolecular G4sdid not block virus adsorption but rather inhibited viral-specifictranscripts. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Ojwanget al. J. Aids 7:560-570 (1994).

Structure-Function Studies

It is known that G-rich nucleic acid sequences can fold, in the presenceof Na⁺ or K⁺ ion, to form orderly structures stabilized by guanosinetetrads. Depending on sequence, intramolecular folds, Rando et al. J.Biol. Chem. 270: 1754-1760, 1995), dimers (Smith, F. W., & Feigon, J.(1992) Nature (London) 344, 410-414, Sundquist, W. I. & Klug, A. (1989)Nature (London) 334, 364-366; Kang, et al. (1992) Nature (London) 356,126131; Balaguumoorthy, P. & Brahmachari, S. K. (1994) J. Biol. Chem.269, 21858-21869), tetrameres (Son, D. & Gilbert, W. (1990) Nature(London) 344, 410-414; Jin, et al. (1990) Science 250, 543-546; Jin, etal. (1992) Proc. Natl. Acad. Sci. USA 89, 8832-8836; Lu et al., (1992)Biochemistry 31, 2455-2459), and higher order associations have beendetected. Such tetrad based structures have been postulated to serve asthe structural basis for telomere function (Sen, D. & Gilbert, W. (1988)Nature (London) 334, 364-366), and have been hypothesized to play a rolein retroviral replication (Bock et al. (1992) Nature (London) 355,564-566), and transcription regulation (Marshall et al. (1992) Proc.Nalt. Acid. Sci. USA 8,9, 6265-6269; Wyatt et al. (1994) Proc. Natl.Acad. Sci. USA 91, 1356-60).

Recently, several groups have shown that compounds which containtetrad-based folds may have activity as potential drug compounds. Bockand colleagues have shown that an intramolecular fold, obtained by aSELEX procedure can bind tightly to thrombin, so as to inhibit clotting(Bock et al. (1992) Nature (London) 355, 564-566). Additionally Wyatt etal. (Wyatt et al. (1994) Proc. Natl. Acad. Sci. USA 91, 1356-60) hasshown that a dimer-wise pairing of phosphorothioate oligomers with thesequence T2G4T2 (four stranded intermolecular tetrads) gives rise toanti-HIV activity, by inhibition of viral adsorption to the cellsurface.

The present inventors have also obtained evidence for sequence-selectiveinhibition of HIV-1 by simple phosphodiester oligonucleotides which formG-tetrad based structures. The highest activity was obtained with a 17mer, referred to as T30177, with composition G12-T5 (Rando et al.,(1994) J. Biol. Chem. 270, 1754-1760; Ojwang, J. et al. (1995) J. Aids7, 560-570), with 2 phosphorothioate linkages (1 at each end) to blockcellular exonuclease activity (Bishop et al. (1996) J. Biol. Chem. 271,5698-5703). NMR evidence was obtained (Rando et al., (1995) J. Biol.Chem. 270, 1754-1760) to suggest that, by reference to similar oligomers(Smith, F. W., & Feigon, J. (1992) Nature (London) 344, 410-414), T30177forms a stable intramolecular fold which is stabilized by a pair ofG-tetrads, connected by three singlestranded loops and a 1-2 base longtail to either side of the fold. Those preliminary studies suggestedthat oligomer folding was coupled to K⁺ ion binding (Rando et al.,(1995) J. Biol. Chem. 270, 1754-1760). Additional studies have suggestedthat T30177 and related derivatives are potent inhibitors of HIV-1integrase, in vitro (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy39, 2426-35).

Pharmacokinetic Studies-Single Dose

Antisense, triple-helix, duplex decoy, and protein-binding (aptamer)oligonucleotides have been shown to have potential as drugs for thetreatment of a variety of human clinical disorders (Stein and Cheng,1993; Marshall and Caruthers, 1993, Science 259: 1564-1570; Chubb andHogan, 1992, Trends in Biotechnology 10: 132-136; Stull and Szoka, 1995,Pharm. Res. 12: 465-483. A number of oligonucleotides have undergonepre-clinical testing, and several are in human clinical trials. Onefinding that has aroused some concern (Black et al., 1994, AntisenseRes. Dev. 4: 299-301) is the observation that total phosphorothioateoligonucleotides cause hemodynamic changes following rapid intravenousadministration. Severe hypotension, leukopenia, complement activation,and death have been reported to occur in primates after rapid infusionsof total phosphorothioate oligonucleotides (Cornish et al., 1993,Pharmacol. Commun. 3: 239-247; Galbraith et al., 1994, Antisense Res.Dev. 4: 201-206). These findings have raised the question of whether thecardiovascular toxicity is a property of phosphorothioateoligonucleotides, or of all oligonucleotides. On the basis of thesefindings, an FDA commentary has recommended that cardiovascularscreening be performed for the pre-clinical safety assessment ofoligonucleotides (Black et al., 1994).

Pharmacokinetic Studies-Repeat Dose

Oligonucleotides have advanced to the stage that they are now consideredas potential therapeutics for the treatment of a variety of humandiseases, and several are presently in clinical trials. Pre-clinicalstudies have generally shown that doses up to approximately 50 mg/kg aresafe, but that higher doses can cause kidney and liver damage, and death(Srinivasan and Iversen, 1995, J. Clin. Lab. Analysis 9:129-137) Bolusintravenous administration has posed a particular concern since it hasbeen shown to sometimes result in serious hypotensive events in primates(Cornish et al., 1993; Galbraith et al., 1994; Black et al., 1995).However, because the number of oligonucleotides that have been studiedhas been small, it is difficult to conclude at the time of making theinvention whether all oligonucleotides share similar toxicities. Inparticular, given the various ways of modifying the backbone ofoligonucleotides (Wu-Pong, 1994, BioPharm 7:20-33) and their ability tofold into distinct three-dimensional structures (Stull and Szoka, 1995,Pharm. Res. 12:465-483), Rando et al. J. Biol. Chem. 270; 1754-1760,1995, the safety profile of different oligonucleotides may be quitedistinct.

Human Clinical Trials

In addition to toxicological studies, efficacy studies should be carriedout for oligonucleotide drugs. In the past, the preferred method oftesting drug efficacy, especially in HIV-1 infected patients, was tomonitor survival of treated patients. However, recent statisticalstudies have shown that a good indicator of anti-HIV drug efficacy isthe reduction in the numbers of copies of viral genome per unit ofpatient serum (viral load). Mellors et al. (1996) Science 272:1167-1170.Reductions in viral load of 90%, or more preferably 99% are desired.However, reductions of viral load of lesser percentages can be useful,especially where the trend of the overall treatment regime isconsistently downward.

Thus, there is a substantial need for antiviral drugs with novelchemistry and with sites of activity distinct from drugs presently used.Most highly desired would be antiviral drugs whose efficacy in humans isknown.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there are provided methodsand compositions useful in treating pathophysiological states caused byviruses, comprising administering a pharmacological dose of anoligonucleotide, the dose being sufficient to inhibit production of thevirus, wherein the oligonucleotide contains a high percentage ofguanosine bases. In preferred embodiments, the oligonucleotide has athree dimensional structure and this structure is stabilized byguanosine tetrads. In a further embodiment, the oligonucleotidecompositions of the invention have two or more runs of two contiguousdeoxyguanosines. In certain embodiments of the present invention, thetarget virus is either herpes simplex virus, human immunodeficiencyvirus, human papilloma virus, human cytomegalovirus, adenovirus, andhepatitis B virus.

In still yet another embodiment of the present invention, there isprovided a guanosine-rich oligonucleotide having a three dimensionalstructure, wherein the three dimensional structure is stabilized byguanosine tetrads or at least two runs of two contiguous deoxyguanosinesand wherein these oligonucleotides exhibit anti-viral activity. In afurther embodiment, the oligonucleotides of the present invention havepartially (pPT) or fully (PT) phosphorothioated internucleoside linkages(backbones) or other chemical modifications. In a further embodiment,the oligonucleotides of the present invention have chemically modifiedor unnatural (synthetic) bases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures for Section A

FIG. 1A shows a 1973 base pair Hind III to Eco R1 sub fragment of theFriend Murine Leukemia Virus (FMLV) clone 57 genome. FIG. 1B shows a 172base pair (HindIII to StuI) fragment which is an expanded portion of the1973 base pair fragment. Within this fragment is the purine rich targetto which triple helix forming oligonucleotides are directed. FIG. 1Cshows the entire Hind III/Eco R1 FMLV fragment cloned into the pT7-2plasmid (United States Biochemical Corporation) yielding p275A. In thisrecombinant the Hind III site is 10 base pairs downstream of the T7 MRNAstart site. The 5′ portion of the triple helix target region is 63 basepairs downstream of the mRNA start and the Dde I site is 131 base pairsdownstream of the mRNA start site. FIG. 1D shows the Hind III/Eco R1FMLV fragment was cloned into pBS (Stratagene) yielding pBSFMLV. TheHind III site, triple helix target site and Dde I site are respectively50, 103 and 171 base pairs downstream from the mRNA start site.

FIG. 2 shows that G-Rich phosphorothioated-oligonucleotides inducedreduction in HSV-2 viral titer. VERO cells infected with HSV-2 weretreated with various concentrations of the indicated drug. The resultsare plotted as percent virus yield relative to VERO cells infected withvirus but not treated with drug (titer=1). The filled square (B106-62)(SEQ. ID. NO. 5) represents a single concentration point (20 μM) forthis oligonucleotide. B106-96 is the fully phosphorothioated version ofB106-62 (SEQ. ID. NO. 5). B106-97 is the fully phosphorothioated versionof B106-71 (SEQ. ID. NO. 6). ACV (4a and 4b) is acyclovir tested againsttwo different stock concentrations of HSV-2 strain HG52. In twoexperiments, after virus infection and before reapplication ofoligonucleotide (BIO-96 or BIO-97), the cells were rinsed with a pH 3buffer in order to remove all virus not yet internalized (96p3 and97p3).

FIG. 3 shows MT-2 cells infected with 0.01 m.o.i. of HIV-1 and thentreated with various concentrations of oligonucleotide or AZT or ddC.The data represents the number of viable cells remaining in the culturedish, i.e., not undergoing virus induced cytopathic effects (CPE). Inthis graph, 100% is the level of CPE occurring in cultures infected withvirus but not treated with any drug.

FIG. 4 shows the culture media taken from NIH3T3 cells chronicallyinfected with FMLV was mixed with various concentrations of I100-51(SEQ. ID. NO. 29) or I100-12 (SEQ. ID. NO. 27) (fully phosphorothioateversion of I100-00 (SEQ. ID. NO. 20)). The mixtures were then assayedfor the presence of viral reverse transcriptase. The data is presentedas a percent of measurable reverse transcriptase in culture medium nottreated with oligonucleotide.

FIGS. 5A, 5B and 5C show the radioabelled (³²P) full-length or truncatedmRNA transcripts were analyzed by polyacrylamide gel electrophoresis,and then quantitated by cutting out the specific transcript andmeasuring the radioactivity in a scintillation counter. FIG. 5A showsthat the reduction in full length transcripts directed by the T7 and T3promoter when I100-51 (SEQ. ID. NO. 29) (anti-parallel triple helixforming oligonucleotide; FMLV2ap) was added. Samples in which nooligonucleotide was added were counted and used as 100% transcriptionreference points. In all other reactions 4×10⁻⁶M of G101-50 (SEQ. ID.NO. 12) (4e-6) was added and where indicated G101-50 plus I100-51 atconcentrations ranging from 2×10⁻⁹ to 2×10⁻⁶M (2e-9 to 2e-6). FIG. 5Bshows the reduction in full length transcript by I100-01 (SEQ. ID. NO.21) (FMLV2p). T7 directed transcripts were treated as in FIG. 5A.G101-50 was added to each reaction except the control (no oligo) with orwithout various concentration of I100-01 or I100-11 (SEQ. ID. NO. 26)(26% G-ctl). FIG. 5C shows the analysis of truncated (63 base pair)transcript.

FIG. 6 shows inhibition of HIV-1 induced syncytia formation four dayspost-infection. SUP T1 cells were infected with HIV-1_(DV) for fourhours and then treated with various concentrations of oligonucleotides.Four days post-infection cells were scored for syncytium formation. Allassays were performed in quadruplicate and the average values used toplot this graph. The legend to the right of the graph indicates thesymbol used for each oligonucleotide tested.

FIG. 7 shows continued suppression of HIV-1 p24 production seven dayspost removal of oligonucleotide. Four days post-infection withHIV-1_(DV), the media from infected cells treated with oligonucleotides(2.5 μM) was removed and replaced with fresh media withoutoligonucleotide. The presence of viral p24 antigen was then assayed 7and 11-days post infection. All samples were assayed in quadruplicateand the average values used to plot this graph. I100-07 (SEQ. ID. NO.24): I100-15 (SEQ. ID. NO. 33); I100-18 (SEQ. ID. NO. 36); I10021 (SEQ.ID. NO. 39). The legend to the right of the graph indicates the symbolused for each oligonucleotide tested.

FIG. 8 shows a Dixon Plot of random oligonucleotide 1232 (SEQ. ID. NO.41) obtained from kinetic analysis of inhibition of HIV-RT with respectto dNTP. The inhibition constant K_(i) was determined by simultaneouslyvarying dNTP (without dATP) concentrations at the same time as inhibitor(oligonucleotide 1232). The K_(i) determination was performed at 0.125mM, 0.25 mM and 0.5 mM dNTP concentrations with constant Primer-Templateconcentration of 0.2 pM. HIV-RT was used at 1 unit in each reaction. Thereported values are the result of simultaneous independent duplicatesdeterminations.

FIG. 9A reveals PBMCs derived from HIV-1 positive patients were mixedwith HIV-1 negative PBMCs in culture medium containing drug I100-15(SEQ. ID. NO. 33). On day 7 the cocultures were washed and resuspendedin fresh medium containing drug. The p24 levels in medium collected onday 7 (before medium change) and day 10 were assayed for p24. FIG. 9BHIV-1 negative PBMCs from two different donors were infected withHIV-1_(DV) and then incubated in the presence of drug for 10 days atwhich time the culture medium was assayed for the presence of p24antigen.

FIGS. 10A and 10B show inhibition of binding of V3 loop specific Mabs toHIV-1 gp120 by phosphorothioate containing oligonucleotides. Matchedsequence oligonucleotides with either phosphodiester (PD) orphosphorothioate (PT) backbones were assayed for their ability toinhibit the interaction of V3 loop specific Mabs with the gp120molecule: SEQ. ID. NOS. 31 (1173) and 32 (1174); SEQ ID. NOS. 24(I100-07) and 39 (I100-21); or SEQ. ID. NOS. 42 (1229) and 43 (1230). Todo this, immobilized gp120 was preincubated with oligonucleotides beforewashing and the addition of Mab NEA 9284 (FIG. 10A) or Mab NEA 9301(FIG. 10B).

FIG. 11 shows a schematic diagram of the HIV-1 genome not drawn toscale. FIG. 11A shows DNA primers. FIG. 11B shows RNA primers.

FIG. 12 shows analysis of DNA (PCR) and RNA (RT-PCR) extracted from SUPT1 cells three days post-infection with HIV-1. (Top Panel). PCR analysisof HIV-1 infected drug treated SUP T1 cell DNA used 0.1 μg of totalextracted DNA for each reaction. In this experiment either AZT, at 0.3μM which is 10 fold over the IC₅₀ value (lane 1) or I100-15 (SEQ. ID.NO. 33) at 5.0 (lane 2) or 0.3 μM (lane 3) were added to SUP T1 cells atthe same time as HIV-1. Lanes 4 (AZT), 5 (5.0 μM I100-15 (SEQ. ID. NO.33)) and 6(0.3 μM I100-15) are the results of DNA samples obtained fromcells in which drug was added 8 hours post-infection. Lanes 8 to 10contain 10, 100 or 1000 ng of DNA extracted from HIV-1 infected controlSUP T1 cells. The band corresponding to 220 bp is the predicted size ofthe internal β-actin control and the 200 bp fragment is the predictedsize for the amplified portion of the HIV-1 genome. The bottom panelcontains RT-PCR analysis of extracted RNA (1 μg/reaction) obtained fromcells treated in an identical fashion as those described in lanes 1-6 ofthe top panel. Lanes 7 and 8 are control HIV-1 infected cell mRNA andlanes 9 and 10 are the results obtained using uninfected untreated SUPT1 cell mRNA.

FIG. 13 shows the results of three oligonucleotides (10⁻⁵M) incubatedwith increasing concentrations (0, 7.5, 15, 30, 60 and 120 mM) of KCl(lanes 1-6 for I100-15 (SEQ. ID. NO. 33), 7-12 for I100-18 (SEQ. ID. NO.36) and 13-18 for Z106-50). The nucleotide markers are poly dT.

FIG. 14 shows a line model for I100-15 (SEQ. ID. NO. 33) folded into anintramolecular tetrad of the Oxytricha class is depicted. The 5′-end ofthe molecule is in the bottom left hand side. The bases (Gs) are stackedon top of each other with the 4 bases in each plane stabilized throughtheir hydrogen bonding with each other and their interaction with the K⁺ion complex in the center of the tetrad.

FIG. 15 displays a one dimensional NMR analysis of a KCl titration andthermal melting parameters for I100-15 (SEQ. ID. NO. 33).

Figures for Section B

FIG. 16. Dose responsive profile for T30177, AZT and ddC. CEM-SS cellswere infected with HIV-1_(RF)(0.01 MOI) and treated with variousconcentrations of each drug for six days at which time the degree ofHIV-1 -induced syncytium formation (cytopathic effect, cpe) wasaddressed. The results shown are the averages of three or moreexperiments with the standard deviations indicated.

FIG. 17. Effect of T30177 on HIV-1 replication in primary macrophages.Primary macrophages were obtained from PBMC preparations and infectedwith HIV-1_(DV) for 24 hours in the presence of the indicated amount ofdrug. Seven days post-infection the intracellular levels of p24 werequantitated using the Coulter p24 antigen capture ELISA kit. The resultsshown are the averages of three or more experiments.

FIG. 18. Effect of time of drug addition on the inhibition profile ofT30177, AZT, and DS5000. MT4 cells, infected with HIV-1_(IIIB) at a MOIof 1, were treated at various times during (time 0) orpost-virus-infection with the test compounds at a concentration 100-foldhigher than their respective IC₅₀ values. Viral p24 levels in theculture medium were monitored 29 hour post-infection. The results shownare the averages of three or more experiments.

FIG. 19. HeLa-CD4-β-galactosidase cell assays. (FIG. 19A).HeLa-CD4-β-galactosidase cells were incubated in medium containing drugfor one hour before virus was added to the culture medium. One hourafter the addition of virus the cells were washed extensively to removeunbound virus and extracellular test material. Forty-eight hourspost-infection the cells were fixed and stained with X-gal. Bluemultinuclear cells were then counted under an inverted microscope (5).(FIG. 19B). HeLa-CD4-β-galactosidase cells were incubated for 1 hour inthe presence of test compound at which time an equal number of HL2/3cells were added to each well. Cells were incubated for 48 hours atwhich time they were fixed, stained with X-gal and counted under aninverted microscope.

FIG. 20. Long term suppression of HIV-1_(IIIB) after treatment ofinfected cell cultures with T30177. (A) MT4 cells were infected with0.01 MOI of HIV-1_(IIIB) and then cultured for 4 days in the presence ofT30177, AZT, DS5000, JM2763 or JM3100 using a concentration of drugequivalent to 100fold over the respective IC₅₀ value. After 4 days thecells were washed extensively and further incubated in drug free medium.The level of viral p24 antigen in the culture medium was monitored atvarious times after removal of drug from the infected cell cultures. Thevalues given are the averages of three or more experiments.

FIGS. 21A-B. Single cycle analysis of viral DNA. CEM-SS cells infectedwith HIV-1_(SKI) at an MOI of 1, were treated with T30177, UC38, CSB orddC at the indicated time post-viral infection. Time 0 indicates thetreatment of cell cultures with drug during virus infection. After 12hours the DNA was extracted from the infected cells and used as atemplate for PCR. The concentration of drug used in each assay isequivalent to 10 to 100-fold over their respective IC2₅₀ values.

FIGS. 22A-D. Analysis of replicated viral DNA. CEM-SS cells wereinfected with HIV-1_(SKI) at an MOI of 1 and then treated with T30177.Eighteen to 20 hour post-infection the low molecular weight Hirt DNA wasanalyzed using PCR primers which would amplify mitochondrial DNA (FIG.22A), early viral synthesized cDNA (FIG. 22C), viral gag cDNA (FIG. 22D)and viral 2-LTR circles (FIG. 22B). The drug concentrations used were0.0, 0.01, 0.1, 1 and 10 μM corresponding to lanes 1 to 5 respectively.The unlabeled lane in each panel contains molecular size marker controlDNA.

Figures for Section C

FIG. 23. Inhibition of HIV-1 integrase 3′-processing and strand transferand HIV-1_(RF) cytopathicity by guanosine quartets. (FIG. 23A) Schematicdiagram showing 3′-processing (3′P, which liberates a GT dinucleotide)and strand transfer (S.T., which results in the insertion of one3′-processed oligonucleotide into another target DNA), with 5′-endlabeled (asterisk), blunt-ended oligonucleotide. (FIG. 23B) Left panel,concentration-response obtained from a typical experiment. The DNAsubstrate (21 mer), 3′-processing product (19 mer), and strand transferproducts (STP) are shown. Lane 1, DNA along; lane 2, with integrase;lanes 3-6, with integrase in the presence of the indicatedconcentrations of T30177. Right panel, graph derived from quantitation(see Materials and Methods) of the dose response in the left panelshowing inhibition of integrase-catalyzed 3′-processing (open squares)and strand transfer (filled squares). (FIG. 23C) Structures of guanosinequartets oligonucleotides. (FIG. 23D) IC₅₀ values for several G4oligonucleotides against both activities of HIV integrase and HIV-1_(RF)in cell culture. Insertions into the parent compound T30177 are shown byan italicized and underlined nucleotide while mutations are designatedby a lower case nucleotide. The guanosines involved in the quartets areshaded and the loops are designated by the corresponding numbers (seeFIG. 23D).

FIGS. 24A-B. Inhibition of strand transfer and 3′-processing activitiesof HIV-1 integrase by the guanosine quartet T30177. (FIG. 24A) Left,schematic diagram depicting the strand transfer assay using theprecleaved oligonucleotide (19 mer substrate). Right Phosphorimagerpicture showing inhibition of strand transfer with T30177. The DNAsubstrate (19 mer) and strand transfer products (STP) are shown. Lane 1,DNA alone; lane 2, plus integrase; lanes 3-6, plus integrase in thepresence of the indicated concentrations of T30177. (FIG. 24B) Left,schematic diagram depicting the 3′-processing assay using theoligonucleotide labeled at the 3′-end with ³²P-cordycepin (*A) (22 mersubstrate). Right, phosphorimager picture showing inhibition of HIV-1integrase-catalyzed 3′-processing with T30177. Lane 1, DNA alone; lane2, with integrase; lanes 3-6, in the presence of the indicatedconcentrations of T30177.

FIGS. 25A-B. Inhibition of the DNA binding activity of HIV-1 integraseby guanosine quartets. DNA binding was measured after UV crosslinking ofreactions in which integrase was preincubated for 30 minutes at 30° C.with the guanosine quartet prior to addition of the DNA substrate. (FIG.25A) Phosphorimager picture showing differential inhibition of DNAbinding with T30177 and T30659. Lane 1, DNA alone (20 nM); lanes 2, 8,and 14, with integrase (200 nM); lanes 3-7, in the presence of theindicated concentrations of T30177; lanes 9-13, in the presence of theindicated concentrations of T30659. The mitigations of proteins of knownmolecular weight are shown to the right of the gel. (FIG. 25B) Graphderived from quantitation of the does response in (FIG. 25A) showinginhibition of integrase binding by T30177 (open squares) but not byT30659 (filled squares).

FIG. 26. Differential activities of T30177 on wild-type and deletionmutants of HIV integrase. (FIG. 26A) Schematic diagram showing the threedomains of HIV-1 integrase. (B) Inhibition of wild-type IN¹⁻²⁸⁸ (opensquares), IN¹⁻²¹² (closed squares), and IN⁵⁰⁻²¹² (open triangles) in thedisintegration assay. (FIG. 26C) Binding of HIV-1 integrase wild-type(IN¹⁻²⁸⁸) and deletion mutants at a final concentration of 1 μM to³²P-end labeled guanosine quartet T30177 at a final concentration of 250nM. The mobility of proteins of known molecular weight (in KDa) areshown to the right of each figure. Lane 1, T30177 alone; lanes 8-9,binding to wild-type, full-length HIV-1 integrase (IN¹⁻²⁸⁸) in thepresence of the indicted metal; lanes 2-3, binding to IN¹⁻²¹² in thepresence of the indicated metal; lanes 6-7, binding to IN⁵⁰⁻²¹² in thepresence of the indicated metal are lanes 4-5, binding to IN⁵⁰⁻²⁸⁸.

FIGS. 27A-B. DNA binding activity of the zinc finger domain of HIV-1integrase. Binding of IN¹⁻⁵⁵ to T30177 or the viral DNA substrate (seeFIG. 23A, 21 mer). Lanes 1, DNA alone (50 nM); lanes 2, IN¹⁻⁵⁵ (2 μM)with no metal; lanes 3, IN¹⁻⁵⁵ with manganese (7.5 mM); lanes 4, IN¹⁻⁵⁵with magnesium (7.5 mM); lanes 5, IN¹⁻⁵⁵ with manganese (7.5 mM) andzinc (4.2 mM); lanes 6, IN¹⁻⁵⁵ with magnesium (7.5 mM) and zinc (4.2mM); lanes 7-10, IN¹⁻⁵⁵ in the presence of the indicated concentrationof zinc alone.

FIGS. 28A-D. Increased binding to and inhibition by guanosine quartetsin magnesium versus manganese. (FIG. 28A) Phosphorimager picture showingDNA binding of wild-type integrase to radiolabeled T30177. Lane 1, DNAalone (27 nM); lanes 2-5; binding of integrase (200 nM) in manganesebuffer to the indicated concentration of T30177; lanes 6-9, binding ofintegrase (200 nM) in magnesium buffer to the indicated concentration ofT30177. The migrations of proteins of known molecular weight are shownto the right of the gel. (FIG. 28B) Structures of T30177 and two analogsin which the internucleotidic linkages have been changed. (FIG. 28C)graph derived from quantitation (see Materials and Methods) of theinhibition of integrase-catalyzed 3′-processing in the presence ofT30177 and analogs in either magnesium or manganese. Inhibition byT30177 (triangles), T30175 (squares), and T30038 (circles is showneither containing magnesium (filled symbols) or manganese (opensymbols). (FIG. 28D) Table showing IC₅₀ values for 3′-processing for theguanosine quartets in buffer containing manganese and magnesium and theratio of these values.

FIGS. 29A-B. Competition of binding to either U5 viral oligonucleotide(see FIG. 23A, 21 mer) (FIG. 29A) or guanosine quartet T30177. (FIG.29B) Lanes 1, DAN alone; lanes 2, with wildtype, full-length HIV-1integrase. Lanes 3-6 in panel (FIG. 29A), with integrase in the presenceof the indicated concentrations of T30177 added after a 5 minutepreincubation with the U5 viral DNA oligonucleotide. Lanes 3-6 in panel(FIG. 29B), with integrase in the presence of the indicatedconcentrations of viral U5 DNA oligonucleotide added after a 5 minutepreincubation with the guanosine quartet T30177.

FIGS. 30A-B. Inhibition of the related retroviral integrases. (FIG. 30A)Inhibition of 3′-processing and strand transfer catalyzed by HIV-1(lanes 2-8), HIV-2 (lanes 9-15), FIV (lanes 16-22), and SIV (lanes23-29) integrases in the presence of T30177. Lane 1, DNA alone; lanes 2,8, 9, 15, 16, 22, 23, and 29, with integrase; lanes 3-7, 10-14, 17-21,and 24-28, with integrase in the presence of the indicatedconcentrations of T30177. (FIG. 30B) Graph derived from quantitation(see Materials and Methods) of the dose responses in (FIG. 30A) showinginhibition of HIV-1 (open rectangles), HIV-2 (filled rectangles), FIV(open triangles), or SIV (filled triangles) integrase-catalyzed3′-processing.

FIG. 31. Three-dimensional drawings of certain guanosine tetrad formingoligonucleotides referred to in Tables C-1 and C-2. Halosubstituted, endmodified, and intermolecular guanosine quartets are shown.

FIG. 32. Three-dimensional drawings of certain guanosine tetrad formingoligonucleotides referred to in Tables C-1 and C-2. Unless otherwisespecified, all oligonucleotides have phosphorothiodiester linkagesbetween the ultimate and penultimate bases at both the 5′ and 3′ ends.(*) denotes the position of the phosphorothiodiester linkages.

FIG. 33. Percentage inhibition of 3′ processing by certainoligonucleotides in Table C-1.

FIG. 34. Inhibition of syncytium formation by certain oligonucleotidesin Table C-1.

FIG. 35. Mutations in the loops of T30177. Three-dimensional drawings ofcertain guanosine tetrad forming oligonucleotides referred to in TablesC-1 and C-2.

FIG. 36. Mutations, deletions and insertions in G quartets.Three-dimensional drawings of certain guanosine tetrad formingoligonucleotides referred to in Tables C-1 and C-2. IC₅₀ for 3′proc./str. tra. is indicated to the right of each tetrad.

Figures for Section D

FIGS. 37A-B. Structure Models. FIG. 37A. The sequence and a structuremodel for oligonucleotides used in this study presented All fouroligomers have been modified so as to include a single phosphorothioatelinkage at the 5′ and 3′ terminus. Proposed sites of G-quartet formationhave been identified by dotted lines. The continuity of thephosphodiester backbone is identified by solid lines. FIG. 37B. A twostep kinetic model for ion induced folding of oligomers in this study.It is proposed that binding a first K⁺ or Rb⁺ ion equivalent, marked asa (+), occurs within the central G-octet, which has been identified bydotted lines. This first step is relatively fast, and is associated withhigher apparent ion binding affinity. It is also associated withformation of unstacked loop domains, and the resultant net loss of UVhypochromism, as compared to the initial random coil state. The secondstep in the process involves as many as two additional K⁺ or Rb⁺ ionequivalents, (+), at the junction between the core octet and flankingloop regions. This second step requires significant ordering of theflanking loop domains, and is therefor associated with an increase ofbase stacking interaction, and a generally high activation energy.

FIGS. 38A-C. Thermal Stability of Oligomer Folding. Thermal denaturationof oligomers has been measured as a function of ion type, ionconcentration and strand concentration. Data have been obtained at 240nm, in 20 mM Li3PO4, pH 7, as the supporting buffer. Tm values werecalculated from the first derivative of a plot of absorbance vs.temperature, but similar values were obtained by using the midpoint ofthe overall absorbance change. FIG. 38A. Tm values for T30695 (curve a),T30177 (curve b), T30376 (curve c), and T30677 (curve d) obtained as afunction of added 12 KCl concentration. FIG. 38B. The Tm Of T30695obtained as a function of KCl, RbCl, NaCl or CsCl concentration. FIG.38C. The strand concentration dependence of Tm has been measured at 1 mMof added KCl.

FIGS. 39A-C. Oligomer Folding Monitored by Circular dichroism (CD). CDdata have been obtained at 25° C. in 20 mM Li3PO4 as a function of addedion concentration. Data have been presented as molar ellipticity inunits of dmole bases. FIG. 39A. The CD spectrum of T30695 in thepresence of 0 mM (curve a), 0.05 mM (curve b), or 10 mM (curve e) ofadded KCl. FIG. 39B. The change in ellipticity at 264 nm, relative tothat measured in the absence of added ion is presented as a function ofadded KCl concentration for T30695 (curve a), T30177 (curve b) andT30676 (curve c). The overall midpoint of the measured KCl inducedtransition has been plotted for each oligomer: 0.02 mM, 0.15 mM and 0.27mM, respectively. FIG. 39C. T30695 has been treated with increasingconcentration of several different cations. The change in ellipticity at264 nm was then measured as described in part B as a function of addedKCl (curve a), RbCl (curve b) or NaCl (curve e).

FIGS. 40A1-3,B1-3. The Kinetics of Ion Induced Folding. Ion was added tooligomers at time zero in the standard 20 mM Li3PO4 assay buffer. Datahave been presented as absorbance (A) vs. time after addition of metalion. FIG. 40A. Kinetics for T30177 were measured at three added KClconcentrations: 0.2 mM (curve a); 1.0 mM (curve b); and 10 mM (curve e).FIG. 40B. Kinetics for T30695 were measured at three added RbC1concentrations: 1.0 mM (curve a), 5.0 mM (curve b) and 10 mM (curve e).For both, the data has been fit to a sum of two exponentials, i.e.A(τ)=A₁exp(-τ/T₁)+A₂exp(-τ/T₂).

Figures for Section E

FIGS. 41A-D. Mean arterial pressure of cynomolgus monkeys pAor to,during and following intravenous administration of AR177 over tenminutes. Blood pressure was continuously monitored via an indwellingfemoral artery catheter. The values are the mean±s.d. of three monkeysat each dose.

FIGS. 42A-D. Neutrophil levels in blood of cynomolgus monkeys prior to,during and following intravenous administration of AR177 over tenminutes. Neutrophil levels were determined pre-dose (−10 minutes), andat 10, 20, 40, 60,120 and 1440 minutes following the initiation of theten-minute infusion of AR177 into cynomolgus monkeys. The values are themean±s.d. of three monkeys at each dose.

FIG. 43. aPTT versus time profile following a ten-minute infusion ofAR177 to cynomolgus monkeys. aPTT was determined before and at varioustime after intravenous infusion of AR177 as described in the Methodssection. aPTT levels returned to baseline by 24 hours in all groups.Certain aPTT values in monkeys at the 20 and 50 mg/kg dose time points,denoted by asterisks, exceeded the upper limit of the assay.

FIG. 44. Complement factor Bb concentration versus time profilefollowing a ten minute infusion of AR177 to cynomolgus monkeys. Bb wasdetermined before and at various times after intravenous infusion ofAR177 as described in the Methods section. Bb levels returned tobaseline by 24 hours in all groups.

FIG. 45. CH50 levels in blood of cynomolgus monkeys prior to, during andfollowing intravenous administration of AR177 over ten minutes. CH50levels were determined pre-dose (−10 minutes), and at 10, 20, 40, 60,120and 1440 minutes following the initiation of the ten-minute infusion ofAR177 into cynomolgus monkeys. The values are the mean of two monkeys inthe saline and 50 mg/kg groups, and three monkeys in the 20 mg/kg group.Data for the third monkey in the saline and 50 mg/kg groups, and for allof the 5 mg/kg group was not available.

FIG. 46. Plasma C_(max) of AR177 in cynomolgus monkeys administeredAR177 as a ten-minute intravenous infusion. The plasma concentration ofAR177 was determined by anion-exchange HPLC as described in the Methodssection.

FIG. 47. AR177 plasma concentration versus time profiles following aten-minute intravenous infusion to cynomolgus monkeys. The plasmaconcentration of AR177 was determined by anion-exchange HPLC asdescribed in the Methods section. The plasma AR177 concentration at 24hours for the 5, 20 and 50 mg/kg groups were <0.020 g/mL for the 5 and20 mg/kg groups, and 0.24±0.42 μ/mL for the 50 mg/kg group.

FIG. 48. The relationship between plasma AR177 and aPTT in cynomolgusmonkeys following a ten-minute intravenous infusion of 5 mg AR177/kg.The plasma concentration of AR177 was determined by anion-exchange HPLCas described in the Methods section. The baseline aPTT level (at 10minutes prior to dosing) was 32.1±4.4 seconds (mean±s.d.).

FIG. 49. The relationship between plasma AR177 and aPTT in cynomolgusmonkeys following a ten-minute intravenous infusion of 20 mg AR177/kg.The plasma concentration of AR177 was determined by anion-exchange HPLCas described in the Methods section. The baseline aPTT level (at 10minutes prior to dosing) was 41.6±6.7 seconds (mean±s.d.). The aPTTvalue in monkeys at the 10 minute time point, denoted by an asterisk,exceeded the upper limit of the assay.

FIG. 50. The relationship between plasma AR177 and aPTT in cynomolgusmonkeys following a ten-minute intravenous infusion of 50 mg AR177/kg.The plasma concentration of AR177 was determined by anion-exchange HPLCas described in the Methods section. The baseline aPU level (at 10minutes prior to dosing) was 33.2±4.8 seconds (mean±s.d.). Certain aPTTvalues in monkeys at the 10 to 120 time points, denoted by asterisks,exceeded the upper limit of the assay.

Figures for Section F

FIG. 51. AR177 plasma concentration after bolus IV dose 1 or 12 versusdose amount in Cynomolgus monkeys. Cynomolgus monkeys were givenintravenous doses of 2.5, 10 or 40 mg/kg/day every other day for a totalof 12 doses. Blood was obtained 5, 30 and 240 minutes following doses 1and 12. The concentration of AR177 in the plasma of every monkey wasdetermined by anion-exchange HPLC as described in the Methods section.There were six monkeys in the 10 and 40 mg/kg groups, and eight monkeysin the 40 mg/kg group. There was a linear relationship between each doseand the plasma concentration that was achieved at each of the samplingtimes.

FIG. 52. AR177 plasma concentration versus time profile following abolus IV injection (dose 12) to Cynomolgus monkeys. Cynomolgus monkeyswere given intravenous doses of 2.5, 10 or 40 mg/kg/day every other dayfor a total of 12 doses. This figure shows the concentration of AR177 inthe plasma 5, 30 and 240 minutes following dose 12. The concentration ofAR177 in the plasma was determined in every monkey by anion-exchangeHPLC as described in the Methods section. There were six monkeys in the2.5 and 10 mg/kg groups, and eight monkeys in the 40 mg/kg group. Therewere no apparent difference between the disappearance of AR177 from theplasma following the 1st (FIG. F-3) and 12th doses.

FIG. 53. The relationship between the plasma AR177 concentration andaPTT in Cynomolgus monkeys following a bolus IV injection of 2.5 mgAR177/kg. Cynomolgus monkeys were given intravenous doses of 2.5mg/kg/day every other day for a total of 12 doses. This figure shows theplasma AR177 concentration versus aPTT levels 5, 30 and 240 minutesfollowing doses 1 and 12. The concentration of AR177 in the plasma wasdetermined in every monkey by anion-exchange HPLC as described in theMethods section. There were six monkeys in the 2.5 mg/kg group. Thebaseline aPTT levels just prior to (pre-dose) doses 1 and 12 were24.1±3.4 seconds and 22.1±2.2. There was no change in the aPTT levels atany of the time points after the 1st or 12th doses of AR177 at 2.5mg/kg.

FIG. 54. The relationship between the plasma AR177 concentration andAPTT in cynomolgus monkeys following a bolus IV injection of 10 mgAR177/kg. Cynomolgus monkeys were given intravenous doses of 10mg/kg/day every other day for a total of 12 doses. This figure shows theplasma AR177 concentration versus aPTT levels 5, 30 and 240 minutesfollowing doses 1 and 12. The concentration of AR177 in the plasma wasdetermined in every monkey by anion-exchange HPLC as described in theMethods section. There were six monkeys in the 10 mg/kg group. Thebaseline aPTT levels just prior to (pre-dose) doses 1 and 12 were23.3±1.8 seconds and 21.6±2.2. There was a close correlation between theaPTT] levels after the 1st or 12th doses of AR177 at 10 mg/kg and theaPTT levels.

FIG. 55. The relationship between the plasma AR177 concentration andaPTT in cynomolgus monkeys following abolus IV injection of 40 mgAR177/kg. Cynomolgus monkeys were given intravenous doses of 10mg/kg/day every other day for a total of 12 doses. This figure shows theplasma AR177 concentration versus aPTT levels 5, 30 and 240 minutesfollowing doses 1 and 12. The concentration of AR177 in the plasma wasdetermined in every monkey by anion-exchange HPLC as described in theMethods section. There were eight monkeys in the 40 mg/kg group. Thebaseline aPTT levels just prior to (pre-dose) doses 1 and 12 were24.8±3.3 seconds and 22.5±2.5. Certain aPTT] values in monkeys at the 20and 50 mg/kg dose time points at five minutes following doses 1 or 12,denoted by asterisks, exceeded the upper limit of the assay. There was aclose correlation between the aPTT levels after the 1st or 12th doses ofAR177 at 40 mg/kg and the aPTT levels.

Figures for Section G

FIG. 56. AR177 pharmacokinetics following a single IV dose of 0.75 mg/kgto humans. Four HIV-positive human patients were administered AR 177 at0.75 mg/kg as a two-hour intravenous (IV) infusion. Blood samples werecollected in EDTA-coated tubes at various time points during andfollowing the IV infusion. Plasma was obtained following low speedcentriguation of the blood. The concentration of AR177 in the plasma wasdetermined using a validated anion-exchange HPLC method.

FIG. 57. AR177 pharmacokinetics following a single IV dose of 1.5 mg/kgto humans. Four HIV-positive human patients were administered AR177 at1.5 mg/kg as a two-hour intravenous (IV) infusion. Blood samples werecollected in EDTA-coated tubes at various time points during andfollowing the IV infusion. Plasma was obtained following low speedcentriguation of the blood. The concentration of AR177 in the plasma wasdetermined using a validated anion-exchange HPLC method.

FIG. 58. AR177 pharmacokinetics following a single IV dose o 3.0 mg/kgto humans. Two HIV-positive human patients were administered AR177 at3.0 mg/kg as a two-hour intravenous (IV) infusion. Blood samples werecollected in EDTA-coated tubes at various time points during andfollowing the IV infusion. Plasma was obtained following low speedcentriguation of the blood. The concentration of AR177 in the plasma wasdetermined using a validated anion-exchange HPLC method.

FIG. 59. AR177 pharmacokinetics following a single IV dose of 0.75, 1.5or 3.0 mg/kg to humans. Ten HIV-positive human patients wereadministered AR177 at 0.75, 1.5 or 3.0 mg/kg as a two-hour intravenous(IV) infusion. Blood samples were collected in EDTa-coated tubes atvarious time points during and following the IV infusion. Plasma wasobtained following low speed centriguation of the blood. Theconcentration of AR177 in the plasma was determined using a validatedanion-exchange HPLC method.

FIG. 60. AR177 T½ and C_(MAX) following single doses to humans.HIV-positive human patients were administered AR177 at 0.75, 1.5 or 3.0mg/kg as a two-hour intravenous infusion. The concentration of AR177 wasdetermined in the plasma using a validated anion-exchange HPLC method.The C_(MAX) (maximal plasma concentration of AR177) and plasma T½(half-life of AR177 in plasma) were determined using PKAnalyst software(Micro Math, Salt Lake City, Utah).

PD. The term “PD” indicates phosphodiester internucleotide linkages inan oligonucleotide.

PT. The term “PT” indicates phosphorothioate internucleotide linkages inan oligonucleotide.

pPT. The term “pPT” indicates both phosphoroyhioate and phosphodiesterinternucleotide linkages in an oligonucleotide.

FIG. 61. AR177 clearance following single doses to humans. HIV-positivehuman patients were administered AR177 at 0.75, 1.5 or 3.0 mg/kg as atwo-hour intravenous infusion. The concentration of AR177 was determinedin the plasma using a validated anion-exchange HPLC method. The plasmaclearance was determined using PKAnalyst software (Micro Math, Salt LakeCity, Utah).

DETAILED DESCRIPTION OF THE INVENTION INDEX TO DETAILED DESCRIPTION OFTHE INVENTION

Definitions

A. General In Vitro Studies

B. Specific In Vitro Studies and In Vitro HIV Inhibition Using T30177

C. Site of Activity Studies-Viral Integrase Inhibition

D. Structure-Function Studies

E. Single Dose Pharmacokinetic Studies

F. Repeat Dose Pharmacokinetic Studies

G. Human Clinical Trials

Definitions

The following terms as defined will be used in the description of theinvention:

OLIGONUCLEOTIDE

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than ten. Its exact size will depend on many factorsincluding the specificity and anti-viral activity of the oligonucleotidefor various viruses. In addition, bases can refer to unnatural(synthetic) bases used in place of an A, C, T or G.

BASE

In referring to “bases” herein, the term includes both thedeoxyribonucleic acids and ribonucleic acids. The followingabbreviations are used. “A” refers to adenine as well as to itsdeoxyribose derivative, “T” refers to thymine, “U” refers to uridine,“G” refers to guanine as well as its deoxyribose derivative, “C” refersto cytosine as well as its deoxyribose derivative. A person havingordinary skill would readily recognize that these bases may be modifiedor derivatized to optimize the methods of the present invention. Inaddition, bases can refer to unnatural (synthetic) bases used in placeof an A, C, T, or G.

INHIBITION

The term “inhibition” of viral replication is meant to include partialand total inhibition of viral replication as well as decreases in therate of viral replication. The inhibitory dose or “therapeutic dose” ofthe compounds in the present invention may be determined by assessingthe effects of the oligonucleotide on viral replication in tissueculture or viral growth in an animal. The amount of oligonucleotideadministered in a therapeutic dose is dependent upon the age, weight,kind of concurrent treatment and nature of the viral condition beingtreated.

PHARMACOLOGICAL DOSE

The term “pharmacological dose” as used herein refers to the dose of anoligonucleotide which causes a pharmacological effect when given to ananimal or human. The pharmacological dose introduced into the animal orhuman to be treated, will provide a sufficient quantity ofoligonucleotide to provide a specific effect, e.g., (1) inhibition ofviral protein or enzymes, (2) inhibition of viral-specific replication,(3) preventing the target site from functioning or (4) damaging theduplex DNA at the specific site or (5) ablating the DNA at the site or(6) inhibiting the transcription/translation of the gene under theregulation of the site being bound or (7) internal inhibition oftranscription or translation of the gene containing the sequence. Oneskilled in the art will readily recognize that the dose will bedependent upon a variety of parameters, including the age, sex, heightand weight of the human or animal to be treated, the organism or genelocation which is to be attacked and the location of the target sequencewithin the organism. Given any set of parameters, one skilled in the artwill be able to readily determine the appropriate dose.

PATHOPHYSIOLOGICAL STATE

The term “pathophysiological state” as used herein refers to anyabnormal, undesirable or life-threatening condition caused directly orindirectly by a virus.

GTO

The term “GTOs” means an oligonucleotide in which there is a highpercentage of deoxyguanosine, or contains two or more segments (runs) oftwo or more deoxyguanosine residues per segment.

GUANOSINE TETRAD

As used herein, the term “guanosine tetrads” refers to the structurethat is formed of eight hydrogen bonds by coordination of the four O⁶atoms of guanine with alkali cations believed to bind to the center of aquadruplex, and by strong stacking interactions. Of particular interestto the I100-15 class of GTO is the structure of the telomere sequencerepeat T₄G₄, first detected in Oxytricha. The oxytricha repeat has beenstudied in oligonucleotides by NMR and by crystallographic methods. SeeSmith et al., Nature, 1992, 356:164-68, and Kang et al., Nature, 1992356:126-31. As predicted from numerous previous physical and biochemicalstudies, both the NMR and crystallographic studies suggest that foldingis mediated by square planar Hoogsteen H-bonding among G-residues, withoverall antiparallel orientation of the four strand equivalentscomprising the tetrad fold. As expected, the crystallography has shownthat the structure is selectively stabilized by tight binding of a smallmonovalent cation to the O⁶ oxygen of guanosine.

The following examples are offered by way of illustration and are notintended to limit the invention in any manner.

A. General In Vitro Studies

The present invention provides methods and compositions for treating apathophysiological state caused by a virus, comprising the step ofadministering a pharmacological dose of an oligonucleotide, the dosebeing sufficient to inhibit the replication of the virus, wherein theoligonucleotide contains sufficient contiguous guanosines so that aguanosine tetrad (inter- or intra-molecular) can form, and the threedimensional structure of the oligonucleotide is stabilized by guanosinetetrads formed at strategic locations. Generally, this method oftreating a virus-induced pathophysiological state may be useful againstany virus. More preferably, the methods of the present invention may beuseful in treating pathophysiological states caused by viruses such asherpes simplex virus, human papilloma virus, Epstein Barr virus, humanimmunodeficiency virus, adenovirus, respiratory syncytial virus,hepatitis B virus, human cytomegalovirus and HTLV I and II.

Generally, the oligonucleotides of the present invention contain apercentage of guanosine bases high enough to ensure anti-viral efficacy.The guanosine is important in forming tetrads which stabilize the threedimensional structure of the oligonucleotides. Thus, theoligonucleotides of the present invention may have any percentage ofguanosine bases which will allow for tetrad formation provided that theoligonucleotide exhibits anti-viral activity. Preferably, theoligonucleotides of the present invention contain two or more segmentsof two or more guanosine bases, and an overall high percentage of G inorder to enable the oligonucleotide to form at least one quanosinetetrad.

Generally, the oligonucleotides of the present invention may be cappedat either the 3′ or the 5′ terminus with a modifier. Preferably, themodifier is selected from the group consisting of polyamine or similarcompounds that confer a net positive charge to the end of the molecule,poly-L-lysine or other similar compounds that enhance uptake of theoligonucleotide, cholesterol or similar lipophilic compounds thatenhance uptake of the oligonucleotide and propanol amine or similaramine groups that enhance stability of the molecule.

The phosphodiester linkage of the oligonucleotides of the presentinvention may be modified to improve the stability or increase theanti-viral activity. For example, a phosphodiester linkage of theoligonucleotide may be modified to a phosphorothioate linkage. Othersuch modifications to the oligonucleotide backbone will be obvious tothose having ordinary skill in this art.

The present invention also provides specific methods of treating viralstates. For example, the present invention provides a method of treatinga pathophysiological state caused by a virus (in preferred embodiments,as specific virus such as, herpes simplex virus, human papilloma virus,Epstein Barr virus, human immunodeficiency virus, adenovirus,respiratory syncytial virus, hepatitis B virus, human cytomegalovirusand HTLV I and II), comprising the step of administering apharmacological dose of an oligonucleotide, the dose being sufficient toinhibit the replication of the virus, wherein the three dimensionalstructure of the oligonucleotide is stabilized by the formation ofguanosine tetrads.

This invention discloses a novel anti-viral technology. The total numberof antiviral mechanisms by which oligonucleotides, and especially G-richoligonucleotides, work is not completely known, although the inventorshave at least narrowed the sites of action as to certain oligonucleotidedrugs as will be seen below. However, in the different virus culturesystems listed above, G-rich oligonucleotides were able to significantlyreduce virus production in each. More importantly, actual human clinicalstudies have demonstrated the efficacy of the drug in reducing viralreplicons in AIDS patients. The present invention is also drawn tooligonucleotides that have three dimensional structures stabilized bythe formation of guanosine tetrads.

The present invention demonstrates poly and/or oligonucleotides inhibitgrowth of HIV-1, HSV1, HSV2, FMLV and HCMV and other viruses if themolecule contains a high percentage of ribo- or deoxyriboguanosine. Therest of the molecule is composed of thymine, cytosine, xanthosine oradenine nucleotides (ribo- or deoxyribo-), their derivatives, or othernatural or synthetic bases. The 5′ and 3′ termini of the oligonucleotidecan have any attachment which may enhance stability, uptake into cells(and cell nuclei) or anti-viral activity. The backbone which connectsthe nucleotides can be the standard phosphodiester linkage or anymodification of this linkage which may improve stability of the moleculeor anti-viral activity of the molecule (such as a phosphorothioatelinkage).

Structural formulas for representative G-rich oligonucleotides disclosedin the instant invention are listed below in Table A-1.

TABLE A-l SEQ ID NO 5(B106-62)5′-gtggtggtggtgttggtggtggtttggggggtgggg-3′ SEQ ID NO 6(B106-71)5′-gtggttggtggtggtgtgtgggtttggggtgggggg-3′ SEQ ID NO 21(I100-01)5′-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3′ SEQ ID NO24(I100-07) 5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′ SEQ IDNO 28(I100-50) 5′-ggtggtggggtggttgttgggggttg-3′ SEQ ID NO 29(I100-51)5′-ggtggtggggtggttgttgggggttgttgggggtgtgtgggtggt-3′ SEQ ID NO26(I100-11) 5′-gatccatgtcagtgacactgcgtagatccgatgatccagtcgatg-3′ SEQ IDNO 12(Gl01-50) 5′-ggtgggtggtttgtgtggttggtgggttt-3′ SEQ ID NO 13(G105-50)5′-ggggggggggtgtgggggggggttgtggtgg-3′ SEQ ID NO 14(G106-50)5′-ggtgggtgggttggggggtgggtgggg-3′ SEQ ID NO 15(G109-50)5′-ggggtttgggtggggggttgggtggttg-3′ SEQ ID NO 16(G110-50)5′-gggtggtggtgttggtgttgtgtg-3′ SEQ ID NO 17(G113-50)5′-ggtgggggggttggtgtgtttg-3′ SEQ ID NO 1(A100-00)5′-tgggtggggtggggtgggggggtgtggggtgtggggtg-3′ SEQ ID NO 2(A100-50)3′-tgggtggggtggggtgggggggtgtggggtgtggggtg-5′ SEQ ID NO 4(A101-00)5′-ggtggtgggggggggtggggtggtggtgggggtgg-3′ SEQ ID NO 18(HIV26ap)5′-gtgtgggggggtggggtggggtgggt-3′ SEQ ID NO 19(HIV26ctl)5′-gggtgggtgggtgggtgggtgggtgg-3′ SEQ ID NO 9(B107-51)5′-ggtggggtggtggtggttggggggggggggt-3′ SEQ ID NO 10(B133-54)5′-ggtggttggggggtggggggg-3′ SEQ ID NO 11(B133-55)5′-gggtggggtggtgggtggggg-3′ SEQ ID NO 20(I100-00)5′-gttgggggttgttggtggggtggtgg-3′ SEQ ID NO 27(I100- 12,PT)5′-gttgggggttgttggtggggtggtgg-3′ SEQ ID NO 22(I100-05)5′-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-CHOL SEQ ID NO23(I100-06) 5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-CHOL SEQ IDNO 25(I100-08) 5′-gttgggggttgttggtggggtggtgg-CHOL SEQ ID NO 35′-gggtgggtgggtgggtgg-3′ SEQ ID NO 30 5′-gggtggttgggtggttgg-3′ SEQ ID NO31(1173) 5′-gggtgggtgggtgggtgg-3′ SEQ ID NO 32(1174,PT)5′-gggtgggtgggtgggtgg-3′ SEQ ID NO 33(I100-15) 5′-gtggtgggtgggtgggt-3′SEQ ID NO 34(I100-16) 5′-gtggtgggtgggtgggtggtgggtggt-3′ SEQ ID NO35(I100-17) 5′-gtggtgggtgggtgggtggtgggtggtggttgtgggt-3′ SEQ ID NO36(I100-18) 5′-ttgtgggtgggtggtg-3′ SEQ ID NO 37(I100-19)5′-tggtgggtggtggttgtgggtgggtggtg-3′ SEQ ID NO 38(I100-20)5′-gtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′ SEQ ID NO 39(I100-21,PT)5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′ SEQ ID NO 40(1231)5′-gatccatgtcagtgacac-3′ SEQ ID NO 41(1232,PT) 5′-gatccatgtcagtgacac-3′SEQ ID NO 42(1229) 5′-cccccccccccccccccc-3′ SEQ ID NO 43(1230,PT)5′-cccccccccccccccccc-3′ SEQ ID NO 44(1198)5′-ttcatttgggaaacccttggaacctgactgactggccgtcgttttac-3′ SEQ ID NO 45(1200)5′-gtaaaacgacggcca-3′ SEQ ID NO 46(I100-25) 5′-gtggtgggtgggtgggg-3′ SEQID NO 47(I100-26) 5′-gtggtgggtgggtggg-3′ SEQ ID NO 48(I100-35)5′-ggtgggtgggtgggt-3′ SEQ ID NO 49(I100-27) 5′-gtggtgggtgggt-3′ SEQ IDNO 50(I100-28) 5′-gtggtgggt-3′ SEQ ID NO 51(I100-30)5′-gtgggtgggtgggt-3′ SEQ ID NO 52(I100-29) 5′-gtgggtgggt-3′

HSV-2 CULTURE ASSAY

In viral yield reduction assays, Vero cells (4×10⁴ cells/tissue culturewell) were incubated with oligonucleotide(s) for 14 hours before theoligonucleotide was removed and virus (HSV-2 strain HG52) was added tothe cells at a multiplicity of infection (m.o.i.) of 0.1 to 1.0 (4×10³to 4×10⁴ PFU). The infection was allowed to proceed for 10 minutes afterwhich the cells are washed and fresh media, containing the sameoligonucleotide was added for an additional 14 hours. Then, the cellswere subjected to a freeze/thaw lysis after which the released virus wastitered.

HIV-1 CULTURE ASSAY

The SUP T1 T lymphoma cell line was infected with HIV-1 strain DV at amultiplicity of infection (m.o.i.) of 0.1 for one hour at 37° C. Afterthe infection, free virus was washed off and the newly infected cellswere plated (5×10⁴ cells) in quadruplicate in 96 well plates that hadbeen prepared with various dilutions of oligonucleotide. The finalconcentration of drug varied between 0.1 and 20 uM. After 3 days ofincubation at 37° C., the plates were scored for the presence ofmultinucleated giant cells (syncytia).

In assays designed to inhibit syncytia formation, a number ofoligonucleotides exhibited anti-HIV-1 activity. The oligonucleotides andtheir IC50 are listed in Table A-2. I100-05 is the same as I100-01 witha cholesterol group attached to the 3′ end via a triglycyl-linker.I100-08 is the same as I100-00 with a cholesterol group attached to the3′ end via a triglycyl-linker. I100-07 was designed as a sequence isomerto I100-01 and I100-06 is the cholesterol derivative of I100-07. A100-00is the same sequence in the opposite orientation to HIB38p (A100-50).I100-07, originally designed as a control for I100-01 to be used inanti-FMLV experiments, was the most efficacious oligonucleotide testedagainst HIV-1.

In other experiments, the HIV-1 strain LAV was used to infect MT-2 cellsat an m.o.i of 0.01. After 7 days, these cells were scored forcytopathic effects (CPE). In anti-HIV-1 assays in which MT-2 cells wereinfected at an m.o.i. of 0.01, several G-Rich oligonucleotides were ableto inhibit viral-induced cytopathic effects with effective dose 50's(IC50s) in the 0.5-1.0 uM range (FIG. 3). The oligonucleotides shown inFIG. 3 were effective in the 0.5 to 1.0 uM range, including A100-00(HIV38p) and A100-50 (HIV38ap), A101-00 (HIV38ctl), HIV-26ctl. Theoligonucleotide HIV-26ap exhibited less efficacy in this assay with anIC50 in the 5 to 10 uM range. In FIG. 3, TE represents buffer alone,i.e., no drug, while AZT and ddC are control drugs.

TABLE A-2 IC₅₀ for oligonucleotides in an anti-HIV-1 syncytia formationassay. G-Rich oligonucleotide IC50 I100-00 3.75 μM I100-01 4.50 μMI100-05 3.25 μM I100-08 3.25 μM 1100-06 0.70 μM I100-07 0.25 μM A100-003.25 μM

FMLV CULTURE ASSAY

Friend Murine Leukemia Virus (FMLV) was grown in a chronically infectedmurine fibroblast cell line (pLRB215) or was propagated in an acuteassay system by infection of NIH3T3 cells. When the chronically infectedcell line was used, pLRB215 cells were split (1×10hu 5) into 24 wellculture dishes and incubated 16 to 20 hours at 37° C. The media was thenremoved and replaced with media containing various concentrations ofoligonucleotide. After 1, 3 or 5 days, culture media was assayed for thepresence of the viral reverse transcriptase enzyme.

In acute assays, NIH3T3 cells were split (1×10⁴) into 96 well dishes andallowed to incubate for 16-20 hours. After incubation, culture media wasremoved and concentrated virus stock (10 ul) was added to each well in100 ul of completed media containing 2 ug/ml polybrene. The virusinfection was allowed to proceed for 18 hours at which time the viruscontaining media was removed and complete media containing variousconcentrations of oligonucleotide was added. After 4 to 7 days, theculture media was assayed for the presence of viral reversetranscriptase.

HCMV CULTURE ASSAY

Human cytomegalovirus was cultured in the human diploid lung fibroblastcell line MRC-5. These cells were split and placed into 24 well culturedishes and preincubated for 24 hours with various concentrations ofoligonucleotide (0.5 to 20 uM) in complete media. The oligonucleotidewas then washed off and virus was added to the cells (approximately 0.1m.o.i.) for 2 hours at 37° C. The virus was then removed and completemedia containing the same concentration of oligonucleotide was added.Cells were then placed at 37° C. for 10-12 days at which time virus inthe culture media was titered using a standard agar overlay procedure.

BACTERIAL T3 AND T7 ASSAYS

In this assay system, a 2 kb fragment (HindIII to EcoR1) of the FMLVvirus (clone 57) was molecularly cloned between the HindIII/EcoR1 sites10 bp downstream of the bacterial T7 promoter (p275A) or 50 bpdownstream of the bacterial T3 promoter (pBSFMLV2). A shematicrepresentation of these two recombinant plasmids can be seen in FIG. 1.Isolated recombinant DNA was then digested with DdeI. Oligonucleotideswere then incubated with the digested DNA and the mixture was subjectedto in-vitro transcription using either the T7 or T3 bacterial enzymes.

REVERSE TRANSCRIPTASE ASSAY

In this assay, reverse transcriptase (either MMLV or FMLV from pLRB215culture media) was incubated with various concentrations ofoligonucleotide and then assayed using the enzyme linked oligonucleotidesorbent assay ELOSA), the ELOSA kit which is commercially available fromNew England Nuclear.

EUKARYOTIC IN VITRO TRANSCRIPTION

In this assay, a recombinant plasmid containing the HSV-1 IE175 promoterfused to the bacterial chloramphenicol acetyltransferase gene (CAT) waslinearized and used as a template for run off transcription studies.Commercially available HeLa cell nuclear extracts or prepared nuclearextracts of HSV-2 infected VERO cell were used.

INHIBITION OF HSV-2 ACTIVITY

The oligonucleotide B106-62 was originally designed to form a triplehelix structure with a portion of the promoter region of the majorimmediate early protein of HSV-2 (IE175). The phosphorothioatederivative of two oligonucleotides were synthesized and tested foranti-viral activity against HSV-2. FIG. 2 shows that the B106-62oligonucleotide at 20 μM was able to reduce viral titers byapproximately 20% whereas the phosphorothioate version (B106-96) reducedvirus by 50% in the submicromolar concentration range. The controloligonucleotide (106-97), the phosphorothioate backbone derivative ofB106-71, was also able to inhibit virus at the same levels as B106-96.Even when an extensive washing procedure at a pH of 3.0 was employed toremove excess virus not internalized during the infection, incubationwith both B106-96 and B106-97 was able to significantly reduce virusyield. Thus, the inventors concluded that the mechanism of anti-viralactivity was not merely a blocking of the adsorption of HSV-2 virions tocells.

FIG. 2 also shows the results of acyclovir in the same molar range asthe oligonucleotides. Acyclovir was tested against two different stocksof HSV-2 strain HG52, as illustrated in FIG. 4.

OLIGONUCLEOTIDE SYNTHESIS

All oligonucleotides used in these examples were synthesized on a DNAsynthesizer (Applied Biosystems, Inc., model 380B or 394), usingstandard phosphoramidite methods. All oligonucleotides were synthesizewith an amino modified 3′-terminal, which resulted in the covalentattachment of a propanolamine group to the 3′-hydroxyl group or resultedin a cholesterol moiety attached to the 3′-terminal via atriglycyl-linker. Oligonucleotides used in this example were capped attheir 3′-terminal with either a propanolamine or a cholesterol moiety toreduce degradation by cellular exonucleases. Phosphorothioate containingoligonucleotides were prepared using the sulfurizing agent TETD orbeaucauge reagent. The 3′-cholesterol modified oligonucleotides wereprepared and purified as described by Vu et al. (in Second InternationalSymposium on Nucleic Acids Chemistry, Sapporo, Japan, 1993).

STABILITY AND TOXICITY

Guanosine-rich oligonucleotides with either full length phosphodiester(PD) or full length phosphorothioate (PT) backbones were stable in theculture media for 4 days, while oligonucleotides consisting of a morerandom composition of nucleotides were rapidly degraded. This indicatesthat the 3′-modified G-rich oligonucleotides with PD backbones werestable against both endonuclease and exonuclease digestion over adefined four day incubation in culture. The concentration ofoligonucleotide needed to reduce cell proliferation by 50% (TC₅₀) ofselected compounds, based on the dye metabolism assay was approximately40 to 50 μM for oligonucleotides with PD backbones and 15 to 40 μM forthose compounds containing a PT backbone. The TC₅₀ for selectedoligonucleotides are presented in Table A-3. Stability and toxicitytests were replaced as described below

TABLE A-3 Guanosine/Thymidine and Control Oligonucleotide SequencesOligo^(a) Length 3′-Modification^(b) Sequence TC₅₀ ^(c) I100-07 45 meramine 5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′ >50 μM I100-0645 mer cholesterol 5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′I100-00 26 mer amine 5′-              gttgggggttgttggtggggtggtgg-3′ 37μM I100-08 26 mer cholesterol5′-              gttgggggttgttggtggggtggtgg-3′ I100-12 26 mer amine(PT)5′-              gttgggggttgttggtggggtggtgg-3′ 18 μM I100-01 45 meramine 5′-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3′ I100-05 45 mercholesterol 5′-ggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3′ A100-0038 mer amine 5′-tgggtggggtggggtgggggggtgtggggtgtggggtg     -3′ 1173 18mer amine 5′-gggtgggtgggtgggtgg                    -3′ I100-11 45 meramine 5′-gatccatgtcagtgacactgcgtagatccgatgatccagtcgatg-3′ 46.5 μM 123118 mer amine 5′-gatccatgtcagtgacac                    -3′ 1229 18 meramine 5′-cccccccccccccccccc                   -3′ ^(a)Alloligonucleotides listed were synthesized with phosphodiester backbonesexcept I100-12 which had phosphorothioate (PT) linkages. ^(b)The cappinggroup at the 3′-end of the oligonucleotides was either a propanolamineor cholestrol moiety. ^(c)Median inhibitory (toxic) concentration intissue culture.

A. Cytotoxicity and Stability Assays.

The cytotoxicity of selected oligonucleotides was assayed using theCellTiter 96™ Aqueous Non-Radioactivity Cell Proliferation Assay(Promega). This is a colormetric method for determining the number ofviable cells in proliferation or chemosensitive assays using a solutionif MTS. Dehydrogenase enzymes found in metabolically active cellsconvert MTS into a formazan product. The SUP T1 cells used in thecytotoxicity assays were in log phase growth at the time of the assay.Cytotoxicity profiles for GTOs with PD backbones such as I100-15 (SEQ.ID. NO. 33) had TC₅₀s (50% cytotoxic concentration) in the range of 30to 50 μM while GTOs with PT backbones such as I100-15 had TC₅s in the 10to 30 μM range. The TC₅₀ for AZT in this assay format was approximately10 μM.

Blockage of the hydroxyl terminus of oligonucleotides has been shown bymany investigators to greatly reduce degradation by cellularexonucleases. Therefore, all oligonucleotides used in these studies weremodified at their 3′-end with either a propanolamine group or acholesterol group. For stability studies, 10 μM of GTOs were incubatedin MEM (GIBCO) supplemented with 10% FBS. Aliquots were taken after 10min, 1 day, 2 days, 3 days and 4 days. The aliquots at each time pointwere immediately extracted twice with 50:50 phenol-chloroform solutionand then precipitated by the addition of ethanol. The recoveredoligonucleotides were 5′-end-labeled using [γ-³²P]ATP and polynucleotidekinase. The integrity of the oligonucleotides was then analyzed on a 20%polyacrylamide gel with 7 M urea. The results indicated that a portionof each GTO with a PD backbone was present in the culture medium forthree to four days while oligonucleotides composed of a more randomassortment of all four nucleotides were rapidly degraded. In addition,positions within PD GTOs where there existed two or more contiguouspyrimidines were more susceptible to endonuclease digestion than regionscontaining purines or alternating purines and pyrimidines.

INHIBITION OF HIV-1 PRODUCTION IN CULTURE ASSAYS

B. Long Term Suppression of Acute HIV-1 Infections in SUP T1 cells

The anti-HIV-1 activity of a series of guanosine/thymidineoligonucleotides (GTOs), with PD backbones, containing differentsequences motifs was tested. As seen in Table A-2, one of the sequencemotifs tested (oligonucleotide I100-07) was 10 fold more active atinhibiting HIV-1 induced syncytium formation than the other motifstested (e.g. I100-00 shown in Table A-1). I100-07 and its derivatives(length and chemical modifications) were further tested for theirability to inhibit virus in a dose-dependent fashion by measurement ofsyncytium formation and viral p24 production.

Briefly, HIV-1_(DV) was used to infect the SUP T1 lymphoblastoid cellline at an m.o.i. of 0.1 TCID₅₀ for one hour at 37° C. prior to washingand resuspension in increasing concentrations of GTOs. The cells (2×10⁴cells/well) were inoculated in triplicate in 200 ul of RPMI 1640containing 10% fetal calf serum. Four days later, the number of syncytiaper well or the level of p24 in the medium was determined. The resultsof these assays are presented in Table A-4. which results indicated thatGTOs with simple PD linkages were capable of inhibiting HIV-1 syncytiaformation and p24 production in culture.

TABLE A4. Guanosine/Thymidine Oligonucleotide Sequences IC50^(b) (μM)Oligo Length linkage^(a) Sequence Syn p24 T.I.^(c) I100 -07 45 mer PD5′-     gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg 0.25 0.55 -21 45mer PT 5′-     gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg 0.225<0.20 >100 -20 38 mer PD5′-          gtgggtgggtggtgggtggtggttgtgggtgggtggtg 1.00 1.00 -19 29 merPD 5′-                tggtgggtggtggttgtgggtgggtggtg 3.75 2.00 -18 16 merPD 5′-                             tgtgggtgggtggtg 3.75 3.00 -17 37 merPD 5′-          gtggtgggtgggtgggtggtgggtggtggttgtgggt 0.30 0.20 -16 27mer PD 5′-                  gtggtgggtgggtgggtggtgggtggt 0.25 0.15 >200-15 17 mer PD 5′-                           gtggtgggtgggtgggt 0.1250.08 >200 -00 26 mer PD 5′-                 gttgggggttgttggtggggtggtgg3.25 ND -12 26 mer PT 5′-                 gttgggggttgttggtggggtggtgg0.225 >0.20 AZT 0.04 0.40 >200 ^(a)The internucleotide backbone linkagesare denoted as PD for phosphodiester and PT for phosphorothioate.^(b)The IC50 valur for the syncytium p24 inhibition assays in uMconcentration. ^(c)T.I. = therapeutic index.

In order to determine the effect of backbone modification on GTOanti-viral activity, the PD backbone in two oligonucleotides sequencesmotifs was replaced with a PT backbone. The phosphorothioate containingoligonucleotides (I100-12 (SEQ. ID. NO. 27)) and I100-21 (SEQ. ID. NO.24)) were then tested for their ability to inhibit HIV-1 inducedsyncytium formation and production of HIV-1 p24 in the SUP T1 acuteassay system (Table A-4). The results from these assays indicated thatthe presence of phosphorothioate for phosphodiester substitutions in theoligonucleotide backbone molecules in the oligonucleotide backbonegreatly enhanced the anti-viral activity of I100-00 (SEQ. ID. NO. 20)(I100-12 (SEQ. ID. NO. 27) but had little if any effect on I100-07 (SEQ.ID. NO. 24) (I100-21 (SEQ. ID. NO. 39) (Table A-4).

It was apparent from the studies that the anti-viral activity of I100-07(SEQ. ID. NO. 24) was maintained when steps were taken to reduce thelength of the molecule to 17 by deleting segments from the 3′-end(I100-15, -16, -17) (SEQ. ID. NOS. 33, 34, 35) but not by deletions fromthe 5′-end (I100-18, -19, -20) (SEQ. ID. NOS. 36, 37, 38). To furtherdetermine to optimal size of the PD oligonucleotide needed for maximalanti-HIV-1 activity, the I100-15 (SEQ. ID. NO. 33) size variants listedin Table A-5 were synthesized and assayed for antiviral activity.

TABLE A-5 Inhibition of HIV-1 Induced Syncytia Using Size variants of1100-15. oligo Sequence IC50 Syn.(uM) I100-15*5′    gtggtgggtgggtgggt    -3′ 0.16 I100-255′    gtggtgggtgggtgggg    -3′ 0.25 I100-26*5′    gtggtgggtgggtggg     -3′ 0. 12 I100-355′     tggtgggtgggtgggt     -3′ 1.75 I100-275′    gtggtgggtgggt        -3′ 4.50 I100-285′    gtggtgggt            -3′ 4.50 I100-305′      gtgggtgggtgggt     -3′  4.50 I100-295′         gtgggtgggt      -3′  >10.00 AZT 0.02 *At 5 uM these compoundssuppressed virus at least 7 days post-removal of drug. All othercompounds at 5 uM were the same AZT 7 days after removal of drug.

The duration of the viral suppression was assayed by changing the mediumin HIV-1 infected cultures containing 2.5 uM of various oligonucleotidesto complete media without added oligonucleotide on day 4 post-viralinfection. The production of viral p24 antigen was then assayed on day 7and day 11 post-infection. The results of this experiment indicated thatthe shorter variants of I100-07 (SEQ. ID. NO. 24) (I100-15 (SEQ. ID. NO.33) and I100-16 (SEQ. ID. NO. 34)) as well as the PT version of thismolecule (I100-21) (SEQ. ID. NO. 39), were capable of totallysuppressing HIV-1 p24 production for at least 7 days after removal ofthe drug from the culture medium (table A-6). This substantial level ofprolonged inhibition was >99% for I100-15 (SEQ. ID. NO. 33), I100-16(SEQ. ID. NO. 34) and I100-21 (SEQ. ID. NO. 39) when compared to the p24antigen levels obtained for untreated HIV-1 infected cells (Table A-6).The quantitation of p24 production relative to untreated HIV-1 infectedSUP T1 cells for all oligonucleotides tested is presented in Table A-6.The presence of sulfur molecules in the backbone of oligonucleotideI100-7 (SEQ. ID. NO. 24) (I100-21 (SEQ. ID. NO. 39)) had a more markedeffect on the reduction of virus seven days after removal of compoundfrom the culture medium than was observed at the four day post-infectionassay point (Table A-5).

TABLE A-6 Detection of HIV-1 p24 Antigen in the Culture Media ofGTO-Treated SUP T1 Cells. Percent p24^(a) Oligonucleotide (2.5 uM) Day4^(b) Day 7 Day 11 Control SUP T1 cells 100.0% 100.0% 100.0% I100-076.0% 15.9% 8.6% I100-21 (PT)^(d) 0.0% 0.0% 0.0% I100-15 0.0% 0.0% 0.0%I100-16 0.0% 0.0% 0.0% I100-18 144.5% 9.7% 5.3% I100-19 208.0% 21.8%15.0% 1100-12 (PT) 0.0% 0.0% 0.0% ^(a)Level of detectable p24 in culturemedium relative to control (infected but untreated SUP T1 cells aftersubtraction of background values. ^(b)Day 4 post-infection culturemedium was replaced with fresh medium without oligonucleotide. ^(c)SUPT1 cells infected with HIV-1 but not treated with oligonucleotides orAZT were used as positive control cells in this experiment. ^(d)1100-21and 1100-12 contain phosphorothioate backbone linkages (PT).

In control experiments, the culture medium from HIV-1 infected SUP T1cells treated with AZT (4 μM) was also replaced on day 4 post-infectionwith drug free media. In these experiments, two days after removal ofAZT from the culture medium the presence of syncytium was observed inthe HIV-1 infected cell cultures and by day 4 all cells were visiblyinfected with HIV-1.

To determine whether the prolonged suppression of HIV-1 was due totoxicity of the oligonucleotides, SUP T1 cells were counted for alltreated samples 7 days after removal of the oligonucleotides from theinfected cell cultures. The results indicated that for cells treatedwith 2.5 μM of drug there was no difference in the number of cells whencompared with control cultures (uninfected, untreated) of SUP T1 cells.

C. Inhibition of HIV expression in patient derived peripheral bloodmononuclear cells (PBMCs)

I100-15 was assessed for activity in PBMC cultures derived from AIDSpatients. Briefly, PHA activated uninfected PBMC's were added to 4PBMC'sderived from patients with HIV infection in the presence of varyingconcentrations of oligonucleotide. Anti-HIV activity was assessed byanalyzing supernatants, collected every three days from these mixedcultures, for the presence of HIV p24. The PHA activated PBMC's weregrown in the presence of 10 units/ml of IL-1 and medium was exchangedevery three days for a period of three weeks. HIV p24 antigen productionwas assayed in drug-treated as compared to untreated control specimens.It should be noted that the results in these experiments (FIGS. 9A-B)observed for AZT were obtained when AZT was used at 12 uM which isroughly 300 fold greater than the IC₅₀ for this compound.

D. In-Vitro inhibition of HIV-1 reverse transcriptase (RT)

The ability of oligonucleotides to inhibit HIV-1 RT in vitro has beenwell documented. Marshall et al. PNAS 1992 89:6265-6269 have described acompetitive interaction at the active site as the mechanism by whichmono- or diphosphorothioate containing oligonucleotides inhibit HIV-1 RTindependent of whether the molecule tested was antisense, a randomsequence or poly SdC.

In order to determine whether I100-15 or its parent molecule, I100-07(or the PT version I100-21), was interacting with HIV-1 RT, the activityof this enzyme was assayed in the presence of various concentrations ofoligonucleotides. A kinetic analysis of the resultant enzyme inhibitionwas conducted to determine the mechanism of inhibition. The GTOsappeared to be inhibiting the RNA dependent DNA polymerase activity ofthe RT enzyme by competitive inhibition at the active site of theenzyme.

The K_(i) value for all of the oligonucleotides tested is presented inTable A-7. The data indicate that for all oligonucleotides tested thepresence of the sulfur group in the backbone greatly enhanced theinteraction between the oligonucleotides and the enzymes. The medianinhibitory dose (ID₅₀) for these oligonucleotides were also calculated(Table A-7). The ID₅₀ results are based on the ability of thesecompounds to inhibit 10 nM of HIV RT.

Short oligonucleotides (18 mers) with PD or PT backbones were assayed todetermine whether the nature of the nucleotide sequence contributed toinhibition of HIV-1 RT in this assay system. Comparison of the effectsof the PD versions of a GTO (1173 or I100-15), poly dC (1229) or arandom nucleotide sequence (1231) suggested that at this length none ofthe sequence motifs inhibited RT (Table A-7). Other 18 mer PD GTOsequence motifs tested yielded similar results. Enzyme inhibitionmonitored by both K₁ and ID₅₀ was observed for the PT versions of thesesame 18 mer oligonucleotides (Table A-7). The degree of enhancement ofobserved enzyme inhibition for all oligonucleotides tested when thesulfur group was present in the backbone, was between one to threeorders of magnitude (Table A-7).

TABLE A-7 In Vitro Inhibition of HIV-1 RT by PD and PT Oligonucleotides.Oligonucleotides Length Linkage^(b) Ki (μM) ID50 (μM) I 100-00 26 PD0.37 5.0 I 100-12 26 PT 0.005 0.015 I 100-07 45 PD 0.137 2.5 I 100-21 45PT 0.001 0.004 I 100-15 17 PD >5.0 >5.0 1173 18 PD >5.0 >5.0 1174 18 PT0.015 0.0154 1229 (poly dC) 18 PD >5.0 >5.0 1230 (poly dC) 18 PT 0.0440.033 1231 (GATC) 18 PD >5.0 >5.0 1232 (GATC) 18 PT 0.56 0.045 ^(a)Eachpair of oligonucleotides contain the same sequence and differ only inthe nature of their backbone linkage. Oligonucleotides 1229 and 1230were poly dC while the 1231 and 1232 oligonucleotides were a randomsequence of all four bases (GATC). ^(b)The backbone modifications aredenoted as PD for phosphodiester and PT for phosphorothioate.

The results from this set of experiments demonstrated that I100-15 isminimally inhibitory to the RNA dependent DNA polymerase activity ofHIV-1 RT. The data also indicated that chemically modifying GTOs, polydC or a random sequence oligonucleotide greatly enhanced the in vitroinhibitory activity of the molecule. Therefore, chemically modifiedoligonucleotides such as the antiviral G-rich molecule describe by Wyattet al. [112] has, by nature, a different set of characteristics fromoligonucleotides with natural PD backbones.

E. Inhibition of the interaction of HIV-1 gp120 with cellular CD4

The outer envelope glycoprotein gp120 of HIV-1 mediates viral attachmentto the cell surface glycoprotein CD4 in the initial phase of HIV-1infection. The effects of both PD and PT modified oligonucleotides onthis interaction were examined using a gp120 capture ELISA kit.

The concentration of the gp120 used in these studies (125 ng/ml) wasdetermined to be within the linear range of the detection assay. Theability of oligonucleotides to inhibit gp120/CD4 interactions by bindingto gp120 was determined by preincubation of the test compounds withsoluble gp120 before addition to the immobilized CD4. The results ofthis experiment (Table A-8) are presented as the concentration ofoligonucleotide needed to reduce by 50% CD4 bound gp120 (ID₅₀ [gp120]).The reciprocal experiment was then performed to measure the ability ofthe oligonucleotides to inhibit these interactions by binding toimmobilized CD4. In this set of experiments I100-00 (SEQ. ID. NO. 20),I100-07 (SEQ. ID. NO. 24) and the PT versions of these twooligonucleotides were capable of preventing the interaction of gp120with immobilized CD4 (ID₅₀ [CD4], Table A-8). For both sequences tested,the PT version of the oligonucleotide had ID₅₀ values which were 50 to100 fold lower than that of the PD version.

A fixed length (18 mer) set of oligonucleotides with either PD or PTbackbones were assayed to determine whether the nature of the nucleotidesequence contributed to inhibition of gp120/CD4 interactions. As wasobserved for the inhibition of HIV-1 RT, the PD versions of thesemolecules had little or no measurable effects on the binding of gp120with CD4. However, the PT versions of these oligonucleotides did yieldmeasurable inhibitory activity. The 18 mer GTO (1174) interruptedgp120/CD4 interactions at approximately 10 fold lower concentrationsthan poly (SdC)₁₈ (1230) while the random sequence 18 mer (1232) had nomeasurable activity (Table A-7).

TABLE A-8 In Vitro Inhibition or HIV-1 gp120 Interaction with CD4 by PDand PT Oligonucleotides. Oligonucleotide Linkage^(a) ID50 [gp 120](μM)ID50[CD4](μM) I 100-00 PD 3.50 18 I 100-12 PT 0.08 0.475 I 100-07 PD0.80 4.25 I 100-21 PT 0.07 0.048 1173 PD >100 >100 1174 PT 0.09 0.451229 (poly dC) PD >100 >100 1230 (poly dC) PT 1.00 3.25 1231 (GATC)PD >100 >50 1232 (GATC) PT >10 >10 ^(a)Each pair of oligonucleotidescontain the same sequence and differ only in the nature of theirbackbone linkage. ^(b)The backbone modifications are denoted as PD forphosphodiester and PT for phosphorothioate.

F. Oligonucleotide interactions with the v3 loop of HIV-1 gp120

It had been reported previously that poly SdC oligonucleotides were ableto bind to the third variable loop domain of HIV-1 gp120 (v3 loop). Thedegree of interaction was reported to be dependent on the length of theoligonucleotide studied, with a rapid decrease in binding affinityobserved for compounds shorter than 18 nucleotides.

It was noted that the detection antibody used to monitor inhibition ofgp120/CD4 interactions in the capture gp120 ELISA KIT (HRP-α-GP120) asdescribed above (Table A-8) recognized an epitope in the gp120 v3 loop(manufacturer's information). For this reason, control experiments wereperformed to determine whether the observed inhibition of gp120/CD4interactions was due in part, or in whole, to interference with theHRP-α-gp120 detection antibody. The results indicated that I100-07 (SEQ.ID. NO. 24) and I173 (SEQ. ID. NO. 31) (PD backbones) did not inhibitthe detection of immobilized gp120. However, the PT oligonucleotidestested (I100-21 (SEQ. ID. NO. 39) and 1174 (SEQ. ID. NO. 32)) were ableto slightly inhibit the detection of gp120 at oligonucleotideconcentrations above 5 μM. This level of inhibition was too small toaccount for the ID₅₀ [gp120] values presented for I100-21(SEQ. ID. NO.39) and 1174 (SEQ. ID. NO. 32) in Table A-8.

Further analysis of oligonucleotide interactions with the v3 loop wasconducted using a v3 loop specific murine Mab, NEA-9284 (FIG. 10). PToligonucleotides were able to inhibit binding of NEA-9284 to gp120. Thepresence of bound gp120 specific Mab was determined using a HRP-labeledgoat-α-mouse antibody. The results of these experiments indicated thatPT oligonucleotides were able to inhibit binding of NEA -9284 to gp120.The ID₅₀ for the most active oligonucleotide (1100-21) (SEQ. ID. NO. 39)was approximately 4 to 7 μM. This concentration is approximately 10 to30 fold higher than the IC₅₀ for this oligonucleotide against HIV-1 inculture (Table A-8). The PD oligonucleotides tested did not inhibit thebinding of any Mab to gp120. Therefore, it was determined to be unlikelythat this was the mechanism by which the PD GTOs such as I100-07 (andhence I100-15) (SEQ. ID. NO. 33) were inhibiting HIV-1.

G. Analysis of HIV-1 RNA and DNA in single cycle assays

Total RNA and DNA were extracted from SUP T1 cells 36 hours afterinfection with 0.1 m.o.i of HIV-1_(DV). In this assay, the infectedcells were treated with I100-15 (SEQ. ID. NO. 33) or AZT at various timepoints before, during or after infection. Harvesting of the infectedcells at 36 hr post-infection allowed for the analysis of approximatelyone round of viral replication. A schematic diagram of the positions ofthe PCR primers used in the DNA and RNA analysis is shown in FIG. 11.

Total extracted DNA was analyzed using a PCR primer set which wouldamplify a 200 bp portion of the viral genome spanning the repeat element(R) into the gag gene. The primer set detected full-length or nearlycompletely synthesized viral DNA. This is the last region of the minusstrand of viral DNA that is synthesized. Thus, for DNA to be detected bythis primer set, two template-switching events have occurred andcontiguous 5′LTR to gag sequences must be present on either the minus orplus strand of DNA.

In the same reaction mixture, a PCR primer set which would amplify a 220bp region of the human β-actin gene was used. The results indicated thatin cells treated with AZT there was a marked decrease in viral DNAsynthesis when the drug was added up to 4 hrs post-infection (data inFIG. 12 shows zero hour and 8 hour time of addition studies). Theeffects of I100-15 on the early rounds of viral DNA synthesis wasminimal.

The results of this experiment indicated that I100-15 did not inhibitvirus entry into the cells because of the detectable levels of viral DNAeven in samples treated with I100-15 (SEQ. ID. NO. 33) at the same timeas virus infection (zero hour addition). Furthermore, it suggested thatI100-15 (SEQ. ID. NO. 33) had a different mechanism of action comparedto AZT.

Additional experiments using alternative PCR primers suggested thatthere may be alterations in the viral DNA synthesis caused by I100-15(SEQ. ID. NO. 33). The observed amplification products, when primersclustered in the U3 region of the virus were used, yielded a bandingpattern which was not predicted and obviously different from theinfected cell control (untreated) and the AZT treated infected cellsamples.

RNA extracted from HIV-1 infected cells was analyzed by RT-PCR. In thisassay, the antisense primer of the PCR primer pairs was used with MMLVRT and extracted mRNA to synthesize cDNA strand. The resultant cDNA wasthen used as a template in PCR reactions. Two RNA primer sets were usedto analyze unspliced (primers r1 and r2) and spliced (primers r1 and r3)HIV-1 transcripts. Predicted sizes of the amplified products were 101 bpand 214 bp for the unspliced and spliced species respectively. The sameβ-action primers used for the analysis of the DNA samples were used ascontrols in this experiment.

The results obtained using primer pair r1 and r3 are depicted in FIG.12. The results of this experiment clearly indicated that a reducedlevel of HIV-1 specific transcript was observed in samples treated withI100-15 (SEQ. ID. NO. 33) in the samples treated with drug at the sametime as virus infection (zero hours). It was also clear that whilesamples treated with AZT had reduced levels of viral cDNA, viral mRNAwas still being produced. The same decrease in HIV-1 specific transcriptwas observed in viral infected cells treated with I100-15 (SEQ. ID. NO.33) when the r1 and r2 primer pair was used (data not shown).

H. Structural analysis of I100-15 and I100-26

I100-07 (SEQ. ID. NO. 24), and its derivative products including I100-15(SEQ. ID. NO. 33) and I100-26 (SEQ. ID. NO. 47), are composed entirelyof deoxyguanosine (G) and deoxythymidine (T). These G-richoligonucleotides were purified using anion exchange reverse phase HPLC.Using this procedure the oligonucleotide is purified in the presence ofsodium ions. Monovalent cations are known to encourage self-associatedstructures for G-rich molecules, all of which involve formation ofG-tetrads. The G-tetrad formation involves the formation of eighthydrogen bonds by coordination of the four O⁶ atoms of guanine withalkali cations believed to bind to the center of a quadruplex, and bystrong stacking interactions. The oligonucleotides purified using anionexchange chromatography then have an opportunity to form inter- orintra-molecular tetrads. The tetrad structure can be strengthened byreplacing the sodium ion with potassium.

I. Nondenaturing gel analysis

I100-15 (SEQ. ID. NO. 33)(17 mer, Table A-5) was analyzed usingnondenaturing polyacrylamide gel electrophoresis. In this experiment,trace concentrations of radiolabeled oligonucleotide (10⁻⁷M) wasincubated with increasing concentrations of cold oligonucleotide (up to10⁻⁵M) before gel analysis in the presence of monovalent cation. Underthe gel conditions used, I100-15 (SEQ. ID. NO. 33) migrated as a uniqueband faster than a random coiled (denatured) 17 mer oligonucleotidewould and it was shown to do so in a concentration independent fashion(data not shown). This was in contrast to I100-18 (SEQ. ID. NO. 36)(16mer, 10 fold less active than I100-15 (SEQ. ID. NO. 33)) which appearedto migrate as multiple species in a concentration dependent fashionunder the same gel conditions (data not shown). The same phenomena wasobserved when 10⁻⁵ oligonucleotide (total cold and radiolabeledoligonucleotide) was incubated with increasing concentration of KCl(FIG. 13). I100-15 (SEQ. ID. NO. 33) migrated as a unique species at allconcentrations of KCl while I100-18 (SEQ. ID. NO. 36) and Z106-50(ggttgggggttggg) migrated as multiple species.

The results from these assay suggested that I100-15 (SEQ. ID. NO. 33)folds into an intramolecular structure while other G-richoligonucleotides such as I100-18 (SEQ. ID. NO. 36) and Z106-S50aggregate into higher order intermolecular structures. It was noted thatthe total phosphorothioate oligonucleotide G-rich compound described byWyatt et al., P.N.A.S. 1994 91:1356-66, with the sequence T₂G₄T₂, wasclaimed to fold into an intermolecular tetrad. Therefore, I100-15 (SEQ.ID. NO. 33) (PD backbone) is structurally and chemically different fromthe oligonucleotide reported (ISIS PT oligonucleotide).

J. Tetrad Structure. Principally due to its role in telomere formation,the structure of four stranded nucleic acid tetrads has been wellstudied

Most eukaryotes possess a repeating G-rich sequence of the form T/C)nGmwhere n=1-4 and m=1-8. Of particular interest to the study of theI100-15 (SEQ. ID. NO. 33) class of GTO was the structure of the telomeresequence repeat T₂G₄, first detected in Oxytricha. The Oxytricha repeathas been studied in oligonucleotides by NMR, Smith et al., Nature 1992,356:164-68, and by crystallographic methods, Kang et al. Nature, 1992,356:126-31. As had been predicted from numerous previous physical andbiochemical studies, both the NMR and crystallographic studies suggestedthat folding is mediated by square planar Hoogsteen H-bonding among Gresidues, with overall antiparallel orientation of the four strandequivalents comprising the tetrad fold. As expected, the crystallographyhas shown that the structure is selectively stabilized by tight bindingof a small monovalent cation to the O⁶ oxygen of guanosine. Butsurprisingly, both NMR and crystallography confirm that the foldedstructure possess alternating syn/anti glycosidic bond angles (asopposed to all anti for most duplex structures).

Feigon and colleagues have used NMR and modelling to deduce thestructure of a 28 base-long oligonucleotide (G₄T₄G₄T₄G₄T₄G₄, Oxy 3.5)which is capable of forming a well-defined all-antiparallelintramolecular tetrad, Smith et al., Nature 1992, 356:164-68. Thepresent inventors postulated that if the GTO I100-15 (SEQ. ID. NO. 33)were to fold to form a stable intramolecular tetrad, its NMR propertieswould be expected to be similar to those of the Oxy 3.5 molecule.

In the folded state, the salient NMR characteristics of theintramolecular Oxy 3.5 tetrad were as follows:

1. Narrow linewidths, indicative of monomer formation only.

2. Induction of well-defined guanosine N1 Hoogsteen imino resonances inthe 11.2 to 11.7 ppm range. The chemical exchange rate of these protonsis very slow, reflective of the high positive cooperativity of tetradfolding and dissociation.

3. Spectral simplicity, indicative of a single predominant foldedstructure, rather than an equilibrium among different folded structures.

4. Intrabase H8-C1′ and interbase H7-C2″ NOE connectivity which demandsa pattern of alternating syn-anti glycosidic bond angle throughout the“tetrad stem” of the folded structure.

K. One dimensional NMR analysis

Displayed in FIG. 14 is a line model for I100-15 (SEQ. ID. NO. 33),folded to form an intramolecular tetrad of the Oxytricha class. From aphysical perspective, the possibility that an intramolecular tetradstructure might form in high KCl or NaCl is not surprising. What wassurprising was the fact that this model proposed a stem regioncomprising a single G-octet and intervening loop regions which were onlytwo bases long.

In order to test the general feasibility of this model, a detailed 3Dmolecular model for a I100-15 (SEQ. ID; NO. 33) was constructed. In sodoing, the inventors assumed that the 8 G's comprising the octet core ofthe structure formed a standard square planar octet, and that glycosidicangles were as in the crystal and NMR structures of the antiparallelOxytricha tetrads, Smith et al., Nature 1992, 356:164-68, and Kang etal., Nature 1992, 356:126-31. Additionally, a single K⁺ ion wasintroduced into the center of the G-octet, with octahedral coordinationto GO₆. Initially, 2 base loop structures were created so as to connectelements of the octet without disruption. Subsequent to this initialpostulation, the structure was subjected to mechanical refinement withfull electrostatics, employing Charmm parameters in Sybyl.

After refinement, it was observed that coordinates of the octet corewere not significantly altered and that backbone parameters within theloop domains were within acceptable energetic limits.

First, the structure was very compact, nearly spherical, with the threeloop regions and the 5′ “GT tail” comprising the surface of the tetradcore. Based upon this structure, it appeared likely that interactionwith cellular macromolecules would be heavily dominated by thestructures of these surface loops. In that regard, the inventors believethat it may be inappropriate to think of such interactions as “tetradbinding.” The inclusion of G-tetrads in such a structure may not beimportant as a recognition element per se, but instead provides alatticework upon which an orderly loop array is positioned.

Further, although the loop regions did not appear to be under mechanicalstress, they were short enough so that they possessed very highconfigurational freedom. Because of those severe length constraints, itwas found that all feasible loop models display a distinct “rabbit ears”structure, wherein the two base planes of the loop region are unstacked,and point outward from the center of the octet core. Such rigid,unstacked, single strand loop character was very distinctive as comparedto other known folded nucleic acid structures. Therefore, varying thesequence or chemical structure of these loops, one at a time, wasnecessary to determine if bonding interactions between these loops andcellular macromolecules are important to the observed anti-HIV activity.

The structures described above possessed a single G-octet core, whichwas known to be the minimum structure required for nucleation of tetradformation. Therefore, when paired with the observed short loop size, theintramolecular tetrad structure proposed for I100-15 (SEQ. ID. NO. 33)is best described as meta-stable, relative to other more robust tetradswhich have been described in the literature. An increase of the corefrom 2 to 3 stacked tetrads, or an increase in the length of flexibilityof one or more loops would be expected to increase the thermodynamicand/or kinetic stability of this structure significantly. Thus, theobserved anti-HIV activity can be improved by sequence modificationwhich enhances the stability of the underlying tetrad latticework.

Finally, it was observed that I100-15 (SEQ. ID. NO. 33) and homologuesdisplay profound resistance to cellular nucleases. One interestingaspect of the proposed structure was that, even in the loop domains,phosphodiester linkages are generally buried from interaction with largesolutes, such as a nuclease. The structure analysis proposed definedlocal phosphodiester backbone structure at low resolution. When pairedwith explicit biochemical analysis of phosphodiester cleavage rate, itis possible to define sites for selective introduction of backbonemodification in I100-15 (SEQ. ID. NO. 33) homologies, for the purpose ofextending the biological half life in vivo.

The gel electrophoresis data described above suggested that I100-15(SEQ. ID. NO. 33) spends very little time as a random coil at 25° C.,under native salt conditions. Although the gel data rules outintermolecular associations, the data do not constrain the oligomer toany particular folded monomeric structure. Oligonucleotide folding inI100-15 (SEQ. ID. NO. 33) has been studied employing a combination ofhigh resolution NMR and methods.

Stable formation of a discrete octet core, mediated by tight binding ofa single monovalent ion is crucial to the model described above. Giventhat G-N1 imino protons give rise to sharp, characteristic ¹H NMRsignals in such a structure, focus has been on the potassium iondependence and temperature dependence of I100-15 (SEQ. ID. NO. 33)folding, as assessed by ¹H NMR at 500 mHz.

For these measurements, I100-15 (SEQ. ID. NO. 33) was synthesized at 15uM scale employing fast deblocking “Expedite” chemistry on a Milligensynthesizer. Subsequent to purification by denaturing anion-exchangechromatography in base (10 mM LiOH, 0.2 to 0.7M NaCl), oligomer puritywas confirmed by denaturing gel electrophoresis (7M urea, 65° C.). ForNMR, the oligomer was desalted and transferred into 20 mM LiC1 adjustedto pH 6.0, which minimizes folding to form tetrads. Oligonucleotidestrand concentration was held constant at 2.7 mM. NMR was measured inH₂₀, employing a Redfield pulse sequence to saturate the waterresonance, as described previously, Dittrich et al., Biochemistry, 199433:4111-4120.

In FIG. 15 a KCl titatation is displayed. At 300° K., in the absence ofadded K⁺, imino proton signals cannot be resolved in the 10-12 ppmregion. Subsequent to addition of KCl, substantial narrowing of iminosignals was obtained, saturating at an added KCl concentration of 3 mM,which is very close to one added K⁺ equivalent per octet. Above 4 mM, itcan be seen that at least two classes of imino resonance can be detectedin the 10-12 ppm range with roughly equal intensity: a broad envelopefrom 10-11 ppm, upon which several sharp resonances are superimposed inthe 11-11.5 ppm region.

By analogy with chemical shifts of other G tetrad structures, theinventors tentatively ascribed the sharp imino signals to the 8Hoogsteen H bonds of the core octet. The broad envelope was ascribed tothe G and T imino resonances contributed by the loop and 5′ terminaldomains. Consistent with published tetrad NMR data, a broad envelop ofsignal was detected at 9 ppm, which most likely results from unusuallyslow exchange of guanosine N2 protons engaged in Hoogsteen pairing.

In order to better distinguish the two classes of imino¹H signal and,additionally, to investigate the gross stability characteristics of thefolded I100-15 structure, thermal melting analysis, at 2.7 mM instrands, 6 mM KCl, 20 mM LiCl, pH 6.0 over the range from 300° K. to345° K. was performed.

Substantial line narrowing of “Hoogsteen” imino proton signals was seenat 310° K., which appears to be accompanied by broadening of the poorlyresolved imino envelope at 10.7 ppm. This caused a narrowing of theplateau above 310° K., giving rise to 7-8 well-resolved imino protons at320° K. By reference to the NMR behavior of the Oxytricia tetrad andother tetrad structures, the formation of 7 to 8 narrow, well-resolvedimino resonances at elevated temperatures strongly suggested that in thepresence of one bound K⁺ ion per octet equivalent, I100-15 folded into adiscreet tetrad structure, stabilized by the 8 Hoogsteen H-bonds of thepresumed octet.

In the range from 330 to 340° K., the imino proton spectrum undergoes anabrupt transition, which is likely to be representative of cooperativeunfolding of the octet. Stability of this kind, accompanied byapparently high thermal cooperativity is very striking indeed, and isgenerally indicative of a single, well-defined folded oligonucleotidestructure.

The origin of the shallow temperature dependence of the spectralparameters, leading to enhanced ¹H resolution at 320° K., remains to bedetermined. It is likely to have resulted from weak intermolecularassociation which occur in the millimolar strand concentration range.This interpretation is born out by preliminary analysis of spectralparameters as a function of strand concentration (not shown).Independent of interpretation, the data suggested that high quality NMRdata may be obtained for exchangeable and non-exchangeable I100-15 (SEQ.ID. NO. 33) protons at 35° C., 20 mM, LiCl, 6 mM KCl and 2 mM in strandequivalents.

INHIBITION OF HCMV ACTIVITY

Several different oligonucleotides reduced HCMV titers in tissueculture. Each of the oligonucleotides contained a different percentageof guanosine residues and a different number of total nucleotides in thepolymer. The results of this assay are depicted in Table A-9. Alloligonucleotides were capable of reducing viral titer in cultureincluding G101-50 (SEQ. ID. NO. 12) which contained only 53% G residues(16 out of 30 total nucleotides). In Table A-9, the length and percentguanosine nucleotides is indicated for each oligonucleotide tested.

TABLE A-9 Oligonucleotide Inhibition of HCMV Activity Viral Yield inplaque forming units (PFU) oligonucleotide (% G) G101-50 G105-50 G106-50G109-50 G113-50 Oligo. (53%) (80%) (78%) (65%) (64%) Conc. 30 mer 31 mer29 mer 29 mer 24 mer None 4.5 × 10³ 4.5 × 10³ 4.5 × 10³ 4.5 × 10³ 4.5 ×10³ PFU PFU PFU PFU PFU 20.0 μM Ø 4.5 × 10¹ 2.5 × 10¹ 8.0 × 10¹ 3.5 ×10¹ PFU PFU PFU PFU 10.0 μM 2.5 × 10¹ 1.8 × 10² 4.0 × 10¹ 4.5 × 10¹ 4.0× 10¹ PFU PFU PFU PFU PFU 1.0 μM 7.0 × 10² 1.9 × 10² 6.0 × 10¹ 1.5 × 10²5.0 × 10² PFU PFU PFU PFU PFU 0.5 μM 8.0 × 10² 2.7 × 10² 1.3 × 10² 3.0 ×10² 5.4 × 10^(2˜) PFU PFU PFU PFU PFU

In NIH3T3 cells chronically infected with FMLV, oligonucleotides(FIG. 1) were capable of inhibiting virus production. However,oligonucleotide controls in this experiment were capable of inhibitingvirus production in culture.

IN VITRO ENZYMATIC ASSAYS

Culture media containing FMLV reverse transcriptase (RT) was mixed withvarious concentrations of I100-51 (SEQ. ID. NO. 29) or I100-12 (SEQ. ID.NO. 27), (the phosphodiester backbone of I100-51 (SEQ. ID. NO. 29)having been modified to a phosphorothioate backbone). Reversetranscriptase was measured as described in the section entitled “ReverseTranscriptase Assay” above. FIG. 4 shows that both oligonucleotides werecapable of inhibiting the RT enzyme. Inhibitory concentrations for 50%reduction in RT activity was between 0.5 to 1 μM for I100-51 and lessthan 0.5 uM for I100-12 (SEQ. ID. NO. 27).

The I100-51 (SEQ. ID. NO. 29)(FMLV2ap), attenuated full lengthtranscription directed by either the T7 or T3 polymerases (FIG. 5A). Ascan be seen in FIG. 1, full length transcripts directed by the T7promoter would be 131 bases long while full length transcripts directedby the T3 promoter would be 171 bases long (position of the Dde I siterelative to the mRNA start site). The sequence isomer of I100-51 (SEQ.ID. NO. 29)(I100-01 (SEQ. ID. NO. 21)=FMLV2p), designed parallel to thetarget strand was also capable of significantly inhibiting transcriptionfrom the T7 promoter (FIG. 5B). However, only the anti-parallel triplehelix forming oligonucleotide FMLV2ap inhibited via attenuation oftranscription as can be seen in the build up of a truncated transcriptin the reaction mix (FIG. 5C). The truncated transcript analyzed in FIG.5C was approximately 63 bases long and matched the predicted sizefragment when p275A was used as a template (T7 promoter). G101-50 (SEQ.ID. NO. 12) (53% G) inhibited T7, but not T3 directed, transcription bya mechanism other than attenuation (FIG. 5A) since no truncatedtranscripts were observed when this oligonucleotide was used alone.I100-11 (SEQ. ID. NO. 26) (26% G) increased the level of specifictranscripts directed by the T7 promoter (FIG. 4).

In experiments designed to monitor inhibition of transcriptioninitiation of the HSV-1 IE175 promoter, using oligonucleotides, bothspecific and control G-Rich oligonucleotides were capable of inhibitingeukaryotic transcription when a HeLa cell extract system was used. Theoligonucleotides used were B133-54 (SEQ. ID. NO. 10); B133-55 (SEQ. ID.NO. 11) and B107-51 (SEQ. ID. NO. 9) as specific inhibitors viapotential triple helix mechanism of action and G101-50 (SEQ. ID. NO. 12)and I100-11 (SEQ. ID. NO. 26) as the low G-content controloligonucleotides.

The experiments described above clearly demonstrated the anti-viralactivity in tissue culture assays for several G-Rich oligonucleotidesagainst HSV-2, HIV-1, HCMV and FMLV. In addition, G-Richoligonucleotides specifically inhibited the bacterial RNA polymeraseenzymes T7 and T3, the FMLV and HIV-1 reverse transcriptase enzyme andeukaryotic RNA polymerase.

B. Specific In Vitro Studies and In Vitro HIV Inhibition Using T30177

As was demonstrated by the inventors in the studies initially conductedas described below, T30177 is an oligonucleotide composed of onlydeoxyguanosine and thymidine, it is 17 nucleotides in length is the samesequence as I10015 (SEQ. ID. NO. 33), and it contains singlephosphorothioate internucleoside linkages at its 5′ and 3′ ends forstability. This oligonucleotide does not share significant primarysequence homology with, or possess any complementary (antisense)sequence motifs to the HIV-1 genome. As shown below, T30177 inhibitedreplication of multiple laboratory strains of HIV-1 in human T-cellslines, peripheral blood lymphocytes, and macrophages. T30177 was alsoshown to be capable of inhibiting multiple clinical isolates of HIV-1and preventing the cytopathic effect of HIV-1 in primaryCD4⁺T-lymphocytes. In assays using human peripheral blood lymphocytesthere was no observable toxicity associated with T30177 at the highestconcentration tested (100 μM), while the median inhibitory concentrationIC₅₀) was determined to be in the 0.1 to 1.0 μM range for the clinicalisolates tested, resulting in a high therapeutic index for this drug. Intemporal studies, the kinetics of addition of T30177 to infected cellcultures indicated that like the known viral adsorption blocking agentsdextran sulfate and Chicago sky blue, T30177 needed to be added to cellsduring, or very soon after, viral infection. However, analysis ofnucleic acids extracted 12 hr-post infection from cells treated withT30177, at the time of virus infection, established the presence ofunintegrated viral cDNA, including circular proviral DNA, in the treatedcells. In vitro analysis of viral enzymes revealed that T30177 was apotent inhibitor of HIV-1 integrase reducing enzymatic activity by 50%at concentrations in the range of 0.01 to 0.10 μM. T30177 was also ableto inhibit viral reverse transcriptase activity, however, the 50%inhibitory value obtained was in the range of 1-10 μM depending upon thetemplate used in the enzymatic assay. No observable inhibition of viralprotease was detected at the highest concentration of T30177 used (10μM). In experiments in which T30177 was removed from infected cellcultures 4 days post-HIV-1 infection, total suppression of virusproduction was observed for more than 27 days. Polymerase chain reactionanalysis of DNA extracted from cells treated in this fashion was unableto detect the presence of viral DNA 11 days after removal of drug fromthe infected cell cultures. The ability of T30177 to inhibit bothlaboratory and clinical isolates of HIV-1 and the experimental datasuggested to the inventors that T30177 represented a novel class ofintegrase inhibitors, indicating that this compound was a viablecandidate against evaluation as a therapeutic agent for HIV-1 in humans.

In the present study the inventors disclose the mechanism by which avariant of I100-15 (SEQ. ID. NO. 33) (T30177) was able to inhibitmultiple HIV-1 laboratory strains in acute and long-term suppressionassays. The data indicated that T30177 is a potent and selectiveinhibitor of HIV-1 via at least two mechanisms. One mechanism involvesinterfering with CD4- and gp120-mediated cell fusion events. However,T30177 is 100-fold less effective in inhibiting gp 120-induced cellfusion events than it is at inhibiting an early event in the viral lifecycle, suggesting a specific point of interdiction distinct from that ofblocking virus/cell interactions. The data also clearly showed thatT30177 is a potent inhibitor of the HIV-1 integrase enzyme in vitro andthat by blocking these events in the viral life cycle T30177 is able tosuppress virus production for prolonged periods after an initial shorttreatment regimen with the drug.

Materials Used in In Vitro HIV Inhibition Studies

Oligonucleotides. The deoxyguanosine-rich and otheroligodeoxynucleotides used in this study were synthesized, purified, andcharacterized as previously reported. Ojwang, et al., J. AIDS 7:560-570(1994); Rando, et al, J. Biol. Chem. 270:1754-1760 (1995). The sequenceand phosphorothioate (PT) pattern of the oligonucleotides used inantiviral assays is shown in Table B-7.

Materials. Zidovudine (3′-azido-3′-deoxythymidine, AZT) and thenucleoside analogs 2′,3′-dideoxyionsine (ddI) and 2′,3′-dideoxycytidine(ddC) were obtained from the AIDS Research and Reference ReagentsProgram, National Institute of Allergy and Infectious Diseases. Dextransulfate (DS5000) was purchased from Sigma, and the bicyclam derivativesJM2763 and JM3100 (De Clereq, et al., Antimicrob. Agents Chemother.38:668-674 (1994)) were obtained from Johnson Matthey (Westchester,Pa.). Chicago sky blue (CSB) was obtained from the Drug Synthesis andChemistry Branch, National Cancer Institute.

Cytotoxicity Analysis. The cytotoxicity of T30177 was assayed asdescribed above. The concentration of drug necessary to give one-quarter(TC₂₅), one-half (TC₅₀) or 95% (TC₉₅) of the maximum inhibition ofgrowth response was then determined. The degree of cell proliferationwas determined according to the manufacturer's instructions.

In other experiments the effect of T30177 on the viability of primaryhuman PBMCs, PBLs and macrophages was determined using the trypan bluedye exclusion technique. Griffiths, B., IRL Press, p. 48 (1992), or bymeasuring the degree of [³H]thymidine or [³]leucine uptake in thesecells (McGrath, M. S., et al. Proc. Natl. Acad. Sci. USA 86:2844-2848(1989)).

Antiviral assays

HIV-1 infection assays using cell lines

Laboratory strains of HIV-1, HIV-2, simian immunodeficiency virus (SIV),or the low passage isolate HIV-1_(DV) (Ojwang, et al., J. AIDS 7:560-570(1994)), were used to infect established cell lines using the indicatedmultiplicity of infection (MOI) of virus, for one hour at 37° C. priorto washing and resuspension in medium containing increasingconcentrations of drug. The infected cells (2×10⁴ cells/well) wereinoculated in triplicate in 200 μl of complete medium which containsRPMI 1640 (Life Technologies) supplemented with 10% FBS, penicillin (50U/mL), streptomycin (50 μg/mL) and L-glutamine, (2 mM). Four to 6 dayspost-infection, drug treated and control wells were analyzed for HIV-1induced cytopathic effects, for the presence of viral reversetranscriptase (RT) or viral p24 antigen in the culture medium. Buckheit,et al., AIDS Research and Human Retroviruses 7:295-302 (1991); Ojwang,et al., J. AIDS 7:560-570 (1994); Rando, et al, J. Biol. Chem.270:1754-1760 (1995). Cytopathic effects (CPE) were monitored by eitherdirect counting of HIV-1 inducted syncytium formation or by stainingcells with the tetrazolium dye XT or MTT. Buckheit, et al., AIDSResearch and Human Retroviruses 7:295-302 (1991). The AZT resistantstrain of HIV-1 (ADP/141) was kindly provided by Dr. Brendan Larder andthe AIDS Directed Programme Reagent Project, Medical Research Council,England.

HIV-1 infection of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from blood ofHIV-1 negative and hepatitis B virus (HBV) negative (healthy) donors byFicoll/Hypaque density gradient centrifugation, cultured as described byGartner and Popovic (Gartner et al., In Techniques in HIV Research, p.59-63 (1990)), then activated with phytohemagglutinin (2 μg/mL) andcultured in RPMI 1640 medium supplemented with 15% fetal bovine serum(FBS) and human recombinant interleukin 2 (IL-2, 30 units/mL). After 3days PBMCs (2×10⁵ cells/well) were infected with various isolates ofHIV-1 at a multiplicity of infection (MOI) of 0.01. After 2 hours at 37°C. cells were washed and treated with various concentrations of T30177or AZT, as described by Buckheit and Swanstrom, id. (1991). The mediumwas changed on day 3 or 4 post-infection and fresh drug was added atthese times. Seven days after infection, HIV-1 replication was analyzedusing the Coulter p24 antigen-capture assay. Assays were performed intriplicate. Data was obtained by spectrophotometric analysis at 40 nmusing a Molecular Devices Vmax plate reader.

HIV-1 infection of PBLs

Human peripheral blood lymphocytes (PBLs) were isolated from blood drawnfrom HIV-1 and HBV seronegative donors. PBLs were isolated byFicoll-Hypaque density gradient centrifugation. The PBLs were suspendedin culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine,20% FBS and 50 μg/mL gentamicin) and the cells counted using the trypanblue exclusion technique. After adjustment of cell density to 1×10⁷cells per mL with culture medium, the suspension was placed in a T-75culture flask and incubated flat at 37° C. in a humidified atmosphere of5% CO₂ for 2 hours. The non-adherent cell population was decanted into asterile disposable flask. Phytohemagglutinin (PHA-P) was added to thePBL suspension at a concentration of 2 μg/mL and the PBl preparation wasthen further incubated at 37° C. for 48 hours. At this time an aliquotof the culture was used for virus infectivity studies. PBLs (5×10⁵cells/well) were infected with HIV-1 isolates at an MOI of 0.2. Thislevel of infection yielded a satisfactory virus control RT activityvalue result at day 7 post-infection (Buckheit, et al., id. (1991)). Twohours post-infection, the cells were separated from the virus bycentriguation, washed twice with culture medium, and suspended inculture medium containing IL-2 at a concentration 30 units/mL and at acell density 2×10⁵ PHA-P-stimulated PBL cells/0.1 mL of culture medium.Seven day post-infection, HIV-1 replication was analyzed using eitherthe RT or p24 assay systems. Data was obtained in the p24 assays byspectrophotometric analysis at 450 nm using a Molecular Devices Vmaxplate reader.

Inhibition of acute infection of primary human macrophages

Human macrophage cultures were established as described by Crow et al.Crowe, et al., AIDS Research and Human Retroviruses 3(2):135-145 (1987).Briefly, PBMC's isolated from HIV-1 and HBV seronegative donors wasallowed to adhere to glass at 37° C. for two hours in calcium andmagnesium free PBS (pH 7.4). The non-adherent cells were aspirated andthe adherent cells were washed three times with cold PBS. The adherentmacrophages were scraped free from the plate, counted, and inoculatedinto 96 well plates at a concentration of 10⁵ cells/well in RPMI 1640medium supplemented with 10% human serum. The macrophages werecultivated in RPMI 1640 with 10% human serum. After incubation overnightat 37° C. the macrophages were infected with HIV-1_(DV) at amultiplicity of infection of 0.1 for 24 hours at 37° C. in the presenceof the indicated amount of drug. Unabsorbed virus was then washed offand the cells were further incubated for 7 days at 37° C. in completemedium supplemented with the indicated amount of drug. On day 7post-infection the adherent macrophages were washed extensively with PBSand lysed with detergent. Cytoplasmic HIV p24 levels were thenquantitated and percent inhibition were calculated and compared tocontrol infected but untreated cells.

Long term suppression studies

Long term suppression assays were performed in MT-4 cells infected withHIV-1_(MB) (MOI of 0.01) using drug concentrations representing 1, 10 or100-fold over the median IC₅₀ value for each compound. Four dayspost-infection, cells were washed twice with phosphate-buffered saline(PBS) and resuspended in complete medium without drug (day 0). Viralbreakthrough was monitored at several time points by measurement ofviral p24 antigen production in the culture medium or the presence ofintracellular viral DNA as described previously, (Rando, et al, J. Biol.Chem. 270:1754-1760 (1995)).

Other viral assays

Respiratory syncytial virus (RSV strain A2), and influenza A (FLUAstrain H3N2) virus assays were performed as described by Wyde et al.(Wyde, et al., Drug. Dev. Res. 28:467-472 (1993)) while Herpes Simplexviruses types 1 and 2 (HSV-1, HSV-2) plaque reduction assays wereperformed as previously described. Lewis, et al, Antimicrob. AgentsChemother. 38:2889-2895 (1994). Vesicular stomatitis virus (VSV),Vaccinia virus, Sindbis virus, Coxsackie virus B4, Polio virus-1, andSemliki forest virus assays were performed as described by De Clercq. DeClereq, E., Antimicrob. Agents Chemother. 28:84-89 (1985). Thearenaviridae assays (Junin and Tacaribe viruses) were performed asdescribed by Andrei and De Clercq. Andrei, et al., Antiviral Res.14:287-299 (1990). Punta Toro virus (ATCC VR-559) and Yellow fever virus(vaccine strain 17D) assays were performed using Vero cells.

Flow cytometric analysis of HIV-1 infected lymphocytes

Seven days post-HIV-1 infection of PBMCs, the infected cell culturemedium was analyzed for HIV-1 production using the p24 antigen-captureassay. In addition, cells from both the drug treated and control wellswere analyzed for CD4 and CD8 antigens by cytofluorometry. Briefly,cells were washed and treated with fluorochrome-labeled monoclonalantibodies to CD4 or CD8 (Becton Dickinson). The cells were washed againand fixed with 2% paraformaldehyde before analysis. Crissman, et al.,Flow Cytometry and Sorting, p. 229-230 (1990) and Crowe et al., AIDSRes. Hum. Retroviruses 3:135-145 (1987).

Single cycle analysis of mHV-1 cDNA

CEM-SS cells (2×10⁶ cells/well) in 0.5 mL of complete medium wereinfected with HIV-1_(SKI) at a MOI of 1.0 for 45 minutes on ice at whichtime complete culture medium (10 mL) was added to the cells. Theinfected cells were then pelleted (1000 RPM for 10 min. at 4° C.),washed twice and aliquoted into a 24-well flat bottom plate (2×10⁵cells/well). The indicated amount of drug was added to the infected cellcultures at various times during or post-infection. The cells wereharvested 12 hours post-infection at which time cell pellets were lysedin 100 μL polymerase chain reaction (PCR) lysis buffer (50 mM KCl, 10 mMTris-HCl (pH8.3), 2.5 mM MgCl₂, 0.1 mg/mL gelatin, 0.45% Nonidet P40,0.45% Tween 20 and 75 μg/mL Proteinase K) at 50° C. for one hourfollowed by 95° C. for 10 minutes. The lysate was stored at −20° C.until use.

PCR analysis of viral cDNA was performed using 10 μL of total celllysate in a 100 μL reaction buffer as previously described (Rando, etal, J. Biol. Chem. 270:1754-1760 (1995)). The primers used were5′-ATAATCCACCTATCCCAG TAGGAGAAAT-3′ and5′-TTTGGTCCTTGTCTTATGTCCAGAATCG-3′ which will amplify a 115 bp segmentof the HIV-1 genome. The cycle conditions used were 95° C. for 10minutes to denature the DNA, followed by 30 cycles of 95° C. for 75seconds, 60° C. for 75 seconds, and a final extension step at 60° C. for10 minutes. Thirty μL of the amplification reaction were mixed with 10ul of τ-³²P-labeled internal probe(5′-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3′), placed at 95° C. for7.5 minutes and then annealed at 55° C. for 15 minutes. The resultantproducts were separated by electrophoresis on a 10% polyacrylamide gel.

Analysis of viral replication

CEM-SS cells (2×10⁷) were infected with HIV-1_(SKI) (MOI of I) for 45minutes at 37° C. with gentle mixing. Following virus attachment, thecells were gently pelleted, washed twice and resuspended in completetissue culture medium. The cells were then divided into aliquots,treated with various concentrations of drug and placed in T75 cultureflasks. The cells were incubated at 37° C. for 18-20 hours and thenharvested by centriguation. To extract nucleic acids for analysis ofHIV-1 integration low- and high-molecular weight DNA were prepared fromHIV-1 infected cells (untreated or treated with increasingconcentrations of drug) according to the protocol originally describedby Hirt (Hirt, B. J., J. Mol. Biol. 26:365-369 (1967)) and modified byGowda et al. Gowda, et al., J. Inmunol. 142:773-780 (1989).

DNA (300 ng), obtained from the low-molecular weight Hirt fractions, wasused as the template in PCR analysis undergoing a 30 cycle amplificationreaction using the conditions described by Steinkasserer et al.(Steinkasserer, et al., J. Virol. 69:814-824 (1995)). PCR primer setsincluded control primers for the amplification of mitochondrial DNA(sense, 5′-GAATGTCTGCACAGCCACTTT-3′; antisense,5′-ATAGAAAGGCTAGGACCAAAC-3′; amplified product, 427 bp); primers for thedetection of early viral transcription events (M667 and AA55 primers asdescribed by Zack et al. (Zack, et al., Cell 61:213-222 (1990)),amplified product, 142 bp); primers for the detection of the viral gaggene (sense, 5′-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3′; antisense,5′-TTTGGTCCTTGTCTTATGTCCAGAATG-3′, amplified product 300 bp); andprimers for the detection of circular proviral DNA (sense,5′-CCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3′; antisense, 5′-CAGTGGGTTCCCTAGTTAGC-3′, amplified product, 536 bp). PCR products wereseparated by agarose gel electrophoresis and visualized by ethidiumbromide staining.

Reverse transcriptase enzyme inhibition assays

Purified recombinant RT (HIV-1_(BH10)) was obtained from the Universityof Alabama, Center for AIDS research. The enzyme assays utilized threedifferent template:primer systems, primed ribosomal RNA, gapped duplexDNA, and poly(rA)p(dT)₁₂₋₁₈ to evaluate the inhibition of HIV-1 RT asdescribed by White et al. (White, et al., Antiviral Res. 16:257-266(1991), and Parker et al. (Parker, et al., J. Biol. Chem. 266:1754-1762(1991)).

Integrase enzyme assays

Purified recombinant HIV-1 integrase enzyme (wild-type) was a generousgift from Dr. R. Craigie, Laboratory of Molecular Biology, NationalInstitute of Diabetes and Digestive and Kidney Diseases. The enzyme(0.25 μM) was preincubated in reaction buffer at 30° C. for 30 minutes.All 3′-processing and strand-transfer reactions were performed asdescribed previously by Fresen et al. (Fresen, et al., Proc. Natl. Acad.Sci. USA 90:2399-2403 (1993)) and Mazumder et al. (Mazumder, et al.,Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)). Enzyme reactions werequenched by the addition of Maxam-Gilbert loading dye, and an aliquotwas electrophoresed on a denaturing 20% polyacrylamide gel. Gels werethen dried and subjected to autoradiography using Kodax XAR-2 film orexposed in a Molecular Dynamics Phospholmager cassette.

Protease assays

HIV-1 protease enzyme (Bachem) was diluted to 166 ug/mL in 50 mM NaOAc,5 mM DTT, 2 mM EDTA, and 10% glycerol (pH 5.0) and stored as 10 ulaliquots at −20° C. HIV protease substrate I (Molecular Probes) wasdiluted to a working concentration of 0.32 nmol/μL. Enzyme (20 μL),substrate (20 μL) and drug (20 μL) were added to each well of amicrotiter plate. Positive and negative controls were evaluated inparallel. Fluorescence was quantitated on a Labsystems Fluoroskan IIusing 355 nm for excitation and 460 nm emission wavelengths at 37° C. attime zero and at 30 minute intervals for 2 hours.

HeLa-CD4-β-galactosidase cell assays

Two different assays using genetically engineered HeLa cells wereperformed as described previously. Buckheit, et al., AIDS Research andHuman Retroviruses 10: 1497-1506 (1994). These assays utilized theHeLa-CD4-LTR-β-galactosidase cell line (Kimpton, et al., J. Virol.66:2232-2239 (1992)), which employ a tat protein-induced transactivationof the β-galactosidase gene driven by the HIV-1 long terminal repeat(LTR). One assay involved infecting the HeLa-CD4LTR-β-galactosidasecells with HIV-1 while the second assay monitored the expression ofβ-galactosidase after incubation of the HeLa-CD4-LTR-β-galactosidasecell with HL2/3 cells. Buckheit, et al., AIDS Research and HumanRetroviruses 10:1497-1506 (1994); Ciminale, et al., AIDS Research andHuman Retroviruses 6:1281-1287(1990). The HL2/3 cells express both theHIV-1 envelope glycoprotein and tat gene product so that co-cultivationof these cells with the HeLa-CD4-LTR-β-galactosidase cells would allowfor CD4- and gp120-mediated cell fusion. The extent of cell fusion canthen be monitored by the degree of tat transactivation of LTR-drivenβ-galactosidase expression. Buckheit, et al., AIDS Research and HumanRetroviruses 10:1497-1506 (1994); Ciminale, et al., AIDS Research andHuman Retroviruses 6:1281-1287 (1990).

Results of the In Vitro HIV Inhibition Studies

As described above, the anti-HIV-1 activity, in cell culture assays ofthe oligonucleotide (I100-15) (SEQ. ID. NO. 33) composed entirely of Gand T was established by the inventors. See also, Ojwang, et al., J.AIDS 7:560-570(1994); Rando, et al, J. Biol. Chem. 270:1754-1760 (1995).I100-15 (SEQ. ID. NO. 33) was found to inhibit HIV-1_(DV) in SUP T1cells with a median inhibitory concentration (IC₅₀) of 0.125 μM. I100-15(SEQ. ID. NO. 33) was synthesized with an unmodified (natural) PDinternucloeside linkage and a propanolamine group attached to the3′-terminus to increase the stability of the oligonucleotide. T30177, amodified variant of I100-15 (SEQ. ID. NO. 33), has the same sequence asI100-15 (SEQ. ID. NO. 33) but contains an hydroxyl moiety at its3′-terminus and a single PT internucleoside linkage at both the 5′- and3′-ends.

Cytotoxidty Assays

The cytotoxicity of T30177 was determined using several different celllines and primary human cells as described above. The TC₂₅, TC₅₀ andTC₉₅ values obtained are shown in Table B-1. The cytotoxicity profileobtained for log phase growing cells was variable depending upon thecell line used, while the slower growing PBMCs, PBLs, and macrophagesall tolerated the compound at concentrations exceeding 100 μM asmonitored using the trypan blue exclusion, [³H]thymidine uptake, or[³H]leucine uptake techniques.

TABLE B-1 Cytotoxicity of T30177 in established cell lines and primarycells. CYTOTOXICITY (μM)^(a) Cell Type TC₂₅ TC₅₀ TC₉₅ Cell Lines^(b)CEM-SS 50.8 ± 3.2   92.0 ± 3.0 >100 MT4 34 ± 4.0   70 ± 7.1 >100 CEMx17410 ± 2.5   50 ± 5.2 >100 MT2 27 ± 3.5 61.2 ± 5.5 >100 AA5 45.66 ± 2.0  94.2 ± 3.1 >100 U937 >100 >100 >100 Vero >100 >100 >100NIH3T3 >100 >100 >100 Primary PBLs >100 >100 >100 humanPBMC >100 >100 >100 cells^(c) Macrophages >100 >100 >100 ^(a)TC₂₅, TC₅₀,and TC₉₅ values are the concentrations of T30177 required to inhibit25%, 50% and 95% of growth (cell lines) or cell survival (primary humancells). ^(b)The cytotoxicity of T30177 in human cell lines wasdetermined using log phase growing cells. ^(c)The cytotoxicity of T30177in primary human cells was determined using trypan blue exclusiontechnique or by measuring the uptake of [³H]thymidine or [³H]leucine onslow growing primary cells.

Inhibition of Viral Replication in Cell Lines

CEM-SS cells were infected with HIV-1_(RF) at an MOI of 0.01 and treatedwith T30177 (pPT variant of SEQ. ID. NO. 33, i.e., having partphosphorothiodiester linkages and part phosphodiester linkages, AZT orddC for six days. In this assay system T30177 inhibited HIV-1_(RF)replication in a dose-dependent manner with an IC₅₀ value of 0.075 μMwhile the control drugs, AZT and ddC, had IC₅₀ values of 0.007 and 0.057μM respectively (FIG. 16). T30177 was then assayed against additionalstrains of HIV-1 in a variety of different cell lines. The results fromthese assays showed that the degree of inhibition observed for eachstrain of HIV-1 analyzed was greatly influenced by the cell line used(Table B-2). In addition, as observed for DS5000, T30177 was inhibitoryfor the AZT-resistant strain of HIV-1 tested (ADP/141) which has fourmutations in its RT gene (67N, 70R, 215F and 219Q).

TABLE B-2 Inhibitory effects of T30177, AZT, and DS500 on viralreplication. IC₅₀(μM)^(a) Virus Cell Line T30177 AZT DS5000^(b) HIV-1strains^(c) SKI CEM-SS 0.025 ± 0.006 0.022 ± 0.0001 — MT2  0.06 ± 0.0010.66 ± 0.005 — RF CEM-SS  0.075 ± 0.0002 0.007 ± 0.0002 — MT2 0.270 ±0.04  0.03 ± 0.005 — MT4 0.037 ± 0.03  — 0.018 ± 0.02 DV SUP T1  0.06 ±0.004 0.03 ± 0.005 — IIIB CEM-SS 2.83 ± 0.17 0.002 ± 0.0003 — MT2 1.94 ±0.12 0.01 ± 0.004 — MT4 0.15 ± 0.02 — 0.034 ± 0.016 SUP T1  0.6 ± 0.060.03 ± 0.006 — AA5 <0.32 <0.003 — ADP/141 MT4 0.27 ± 0.05 — 0.032 ±0.008 HIV-2/SIV strains HIV-2_(ROD) MT4 27.5 ± 11.6 — 0.082 ± 0.088HIV-2_(EHO) MT4 5.98 ± 1.05 — 0.084 ± 0.086 SIV_(MAC251) MT4 1.5 ± 1.2 —0.548 ± 0.48 ^(a)The IC50 value is the concentration of drug required toinhibit virus production by 50%. The results presented are the averagesof three or more experiments. ^(b)For DS5000 the μM units are anapproximation based upon the average molecular weight (5000) of thematerial used in these studies.

T30177 was also tested for its ability to inhibit laboratory strains ofHIV-2 and SIV. The results (Table B-2) from these assays indicate thatT30177 is more active against the strains of HIV-1 and SIV tested thanagainst the two strains of HIV-2 tested (ROD and EHO). In addition,T30177 was found to be inactive against a variety of enveloped andnonenveloped viruses tested (Table B-3) with IC₅₀ values found to begrater than the highest concentration of drug tested (200 μg/mL or 37μM). This is in contrast to DS5000 which was found to be a potentinhibitor of all of the enveloped viruses tested except Vaccinia andSemliki forest viruses (Table B-3).

TABLE B-3 Inhibition of viral replication in cell lines treated withT30177 or DS5000. IC₅₀(μg/mL)^(a) T30177 DS5000 T30177 DS5000^(b)Envelope Viruses: HSV-1 (KOS) >200 2 400 >400 HSV-2 (G) >200 2 400 >400HSV-1 TK (B2006) >200 2 400 >400 HSV-1 TK (VMW1837) >200 2 400 >400Sindbis virus >200 10 ≧200 >400 Semliki forest virus >200 >400 ≧200 >400Vesicular Stomatitis virus >200 20 400 >400 Vaccinia virus >200 >400400 >400 Punta Toro virus >200 10.9 >200 >400 Yellow Fever virus >20026 >200 >200 RSV (A2) >200 4 >400 >200 Influenza A (H3N2) >125— >200 >400 Junin virus >50 13 >50 >200 Tacaribe >50 13.5 >50 >200 NonEnveloped Viruses: Coxsackle virus (B4) >200 >400 ≧400 >400 Poliovirus-1 >200 >400 ≧400 >400 ^(a)Concentration of drug required to reducevirus-induced cytopathogenicity by 50% (IC₅₀). The assay results arepresented in μg/mL units. For T30177 5.4 μg/mL is equal to 1 μM and forDS500O 5 μg/mL is approximately equal to 1 μM. ^(b)The minimumconcentration required to cause microscopically detectable alterationsis normal cell morphology (MCC). The results presented are the averagesof the three or more experiments.

Inhibition of HIV-1 Replication in Peripheral Blood Cells

The primary targets of HIV-1 infection in vivo are CD4⁺ T lymphocytesand macrophages. Therefore in the following set of experiments theinventors tested the efficacy of T30177 on HIV-1 replication in PBMCs,PBLs and macrophages.

Activated PBMCs were infected with laboratory strains of HIV-1 andcultured in the presence of T30177, AZT or ddl. Treatment of infectedPBMCs with T30177 inhibited the replication of the all four HIV-1isolates tested with IC₅₀ values ranging from 0.12 to 1.35 μM (TableB-4). In this assay AZT was more efficacious against all HIV-1 isolatestested, on a molar scale, than T30177 while at the same time T30177 wasmore potent than ddI against the two HIV-1 strains tested. It is alsointeresting to note that HIV-1_(IIIB) was more susceptible to T30177 inassays performed using PBMCs than in assays using T-cell lines (TablesB-2 and B-4).

TABLE B4 HIV-1 replication in primary human cells treated with T30177,ddI or AZT Virus Strains IC₅₀ μM^(a) (Cells) HIV-1 Isolate T30177 ddIAZT Laboratory IIIB 0.12 ± 0.006 0.74 ± 0.05 0.003 ± Isolates^(b) 0.0002(PBMCs) JR_(CSF) 0.28 ± 0.04  2.0 ± 0.5 0.0025 ± 0.001  RF 0.75 ± 0.13ND^(C) 0.272 ± 0.003  MN 1.35 ± 0.10 ND 0.053 ± 0.001  Clinical WEJO(SI)0.30 ± 0.01 2.18 ± 0.026 0.017 ± Isolates^(b) 0.0001 (PBLs) BAKI(SI)0.23 ± 0.005 2.61 ± 0.003 0.020 ± 0.006 WOME(SI) 0.71 ± 0.002 0.41 ±0.008 0.025 ± 0.0003 ROJO(SI)  3.9 ± 0.02 0.87 ± 0.001 0.052 ± 0.0004JOGA(NSI) 0.33 ± 0.004 ND >1.0 BLCH(NSI) 3.08 ± 0.006 ND 0.022 ± 0.0008VIHU(NSI)  1.3 ± 0.02 1.21 ± 0.009 0.036 ± 0.0007 S. E. Asia 0.58 ±0.003 ND 0.06 ± 0.005 N. Amer. #1 0.25 ± 0.003 ND 0.01 ± 0.004 N. Amer.#2 2.92 ± 0.005 ND 1.65 ± 0.007 942716 0.86 ± 0.006 ND 0.002 ± 0.003942751 0.38 ± 0.003  2.2 ± 0.02 0.028 ± 0.0025 ^(a)Concentration of drugrequired to inhibit viral production by 50% (IC₅₀) was determined usingthe Coulter p24 antigen capture or RT assays. ^(b)Antiviral assays wereperformed using laboratory strains of HIV-1 in peripheral bloodmononuclear cells (PBMCs) or using syncytium inducing (SI) ornon-syncytium inducing (NSI) clinical isolates of HIV-1 in PBLs. ^(c)Thevalue was not determined (ND).

The therapeutic potential of any anti-HIV drug is dependent upon itsability to inhibit clinical isolates of the virus obtained fromdifferent geographical locations. Therefore, the inventors evaluated theability of T30177 to inhibit the infection of PBLs using a variety ofclinical isolates of HIV-1 which were both syncytium inducing (SI) andnon syncytium inducing (NSI) strains of HIV-1. In addition, the isolatesused in this study had their origins in different geographic regions.After infection with HIV-1 the PBLs were cultured in the presence ofT30177, AZT or ddI for seven days. T30177 inhibited the viralreplication of all the HIV-1 isolates tested with IC₅₀ values rangingfrom 0.23 to 3.08 μM (Table B-4). In the same assay, AZT and ddI hadIC₅₀ values ranging from 0.01 to 1.65 μM and 0.41 to 2.61 μM,respectively. It is important to note that T30177 was active againstboth NSI and SI isolates and was very active against he JOGA isolatewhich was obtained from a pediatric patient. The JOGA isolate was alsoobserved to be relatively resistant to AZT treatment (Table B-4).

Another major target cell of HIV-1 infection is the macrophage. Fullydifferentiated macrophages were infected with HIV-1_(DV) and treatedwith T30177 or AZT. T30177 significantly inhibited HIV-1 replication inmacrophages (FIG. 17). However, due to the long exposure of cells tovirus (24 hours), T30177 and AZT worked best when administered atconcentrations above the IC₅₀ values obtained for these drugs in assaysperformed in established cell lines.

Variations in Viral MOI

To investigate the effect of variations in the MOI on the anti-HIV-1activity of T30177, CEM-SS or MT4 cells were infected with various MOIsof HIV-1_(RF) or HIV-1_(IIIB) (Table B-5). Unlike AZT, T30177 was muchless sensitive to changes in the viral MOI. For example in these assayswhen the MOI of HIV-1_(RF) was changed from 0.01 to 1.28, T30177 onlyexhibited a 14-fold increase in its IC₅₀ value while at the same timethe IC₅₀ value for AZT increased over 1000-fold (Table B-5).

TABLE B-5 Effect of changes in viral multiplicity of infection (MOI) onthe anti-HIV-1 activity of T30177 and AZT. Multi- plicity HIV-1^(b) ofIC50/IC90 in Isolate/ Infec- 23 μM_(a) Cell tion T30177 AZT RF 0.010.20/0.50 ± 0.01/0.03 0.01/0.19 ± 0.001/0.001 (CEM- 0.02 0.41/1.50 ±0.03/0.04 0.02/0.47 ± 0.009/0.046 SS) 0.04 0.60/1.56 ± 0.01/0.020.07/0.86 ± 0.005/0.03  0.08 0.70/1.56 ± 0.01/0.08 0.50/1.0 ± 0.01/0.0050.16 0.87/1.6 ± 0.01/0.03 0.6/>10.0 ± 0.05    0.32  1.25/4.7 ± 0.15/0.278.5/>10.0 0.64 2.64/4.75 ± 0.05/0.16 >10.0/>10.0 1.28 2.81/4.77 ±0.04/0.06 >10.0/>10.0 IIIB 0.02  3.1/6.6 ± 0.23/0.8 0.037/0.22 ±0.003     (MT4) 0.01  2.7/9.2 ± 0.03/0.25 0.01/0.03 ± 0.002    0.3 3.38/7 ± 0.15/0.5 0.15/3.3 ± 0.01/0.05  1  6.8/26 ± 0.53/5.1 0.42/3412± 0.1/10    ^(a)The concentration of drug needed to limit virusproduction by 50 (IC50) and 90 (IC90) percent as measured in the cpeassay ^(b)The strain of HIV-1 and cell line used for each assay isindicated.

Effect of T30177 on CD4 and CD8 T-cell Subsets

One of the principal immunological markers correlated with progressionto AIDS is the decline in T lymphocytes which express the CD4 celldetermining marker (CD4). The change in CD4⁺ T-lymphocytes is usuallymonitored by noting changes in the ratio of CD4⁺ to CD8⁺ lymphocytes inthe blood. To determine the effect of T30177 treatment on the CD4/CD8ratio, CD4 and CD8 antigen expression was analyzed on the surface ofcultured PBMCs seven days post-infection with either laboratory strainsor clinical isolates of HIV-1. In these experiments treatment witheither AZT or T30177 increased the number of CD4⁺ T-cells in the cellculture, relative to untreated infected cultures (Table B-6). Theobserved increase in CD4⁺ cells was dependent on the drug concentrationused and was inversely correlated with the level of virus production(FIGS. 16 and Tables B-2 and B-4). These results suggest that theblockage of HIV-1 replication parallels the suppression of thecytopathic effects of the virus in primary human lymphocytes.

TABLE B-6 Effect of T30177 or AZT on the ratio of CD4/CD8 lymphocytes inHIV-1 infected PBMCs^(a). HIV-1 CD4/CD8 HIV-1 CD4/CD8 Strains Drug Conc.% CD4 % CD8 Ratio Isolates Drug Conc. % CD4 % CD8 Ratio No Virus NoDrug, Day 0 39% 80% 0.49 No Virus No Drug, Day 0 39% 80% 0.49 No Drug,Day 7 45% 71% 0.63 No Drug, Day 7 45% 71% .063 IIIB No Drug  1% 98% 0.01SE Asia No Drug  2% 99% 0.02 0.1 μM AZT 31% 79% 0.39 0.1 μM AZT 29% 82%0.35 1.0 μM AZT 30% 81% 0.37 1.0 μM AZT 31% 82% 0.38 5.0 μM AZT 27% 86%0.31 5.0 μM AZT 29% 86% 0.34 0.1 μM T30177  1% 98% 0.01 0.1 μM T30177 2% 99% 0.02 1.0 μM T30177 18% 85% 0.21 1.0 μM T30177 12% 90% 0.13 5.0μM T30177 27% 83% 0.33 5.0 μM T30177 29% 82% 0.35 10.0 μM T30177  27%83% 0.33 10.0 μM T30177  29% 82% 0.35 MN No Drug  6% 96% 0.06 N. Amer.#1 No Drug 28% 79% 0.35 0.1 μM AZT 25% 82% 0.30 0.1 μM AZT 25% 83% 0.301.0 μM AZT 35% 79% 0.44 1.0 μM AZT 25% 84% 0.30 5.0 μM AZT 36% 78% 0.465.0 μM AZT 26% 85% 0.31 0.1 μM T30177  5% 95% 0.05 0.1 μM T30177 21% 84%0.25 1.0 μM T30177 18% 86% 0.21 1.0 μM T30177 26% 80% 0.33 5.0 μM T3017725% 81% 0.31 5.0 μM T30177 27% 81% 0.33 10.0 μM T30177  26% 80% 0.3310.0 μM T30177  29% 81% 0.36 RF No Drug 14% 89% 0.16 N. Amer. #2 No Drug 4% 97% 0.04 0.1 μM AZT 23% 84% 0.27 0.1 μM AZT  6% 95% 0.06 1.0 μM AZT32% 76% 0.42 1.0 μM AZT 13% 89% 0.15 5.0 μM AZT 34% 81% 0.42 5.0 μM AZT25% 85% 0.29 0.1 μM T30177 10% 92% 0.11 0.1 μM T30177  4% 98% 0.04 1.0μM T30177 26% 82% 0.32 1.0 μM T30177  5% 95% 0.05 5.0 μM T30177 31% 83%0.37 5.0 μM T30177 28% 82% 0.34 10.0 μM T30177  29% 85% 0.34 10.0 μMT30177  27% 84% 0.32 ^(a)The percentage of CD4 and CD8 antigen bearingT-cells in the HIV-1 infected PBMC population was determined by flowcytometric analysis of cells treated with fluorescein labeled a-CD4 ora-CD8 monoclonal antibodies.

In vitro some HIV-1 isolates infect CD4⁺ lymphocytes, shed infectiousvirus into the culture medium but do not cause destruction of theinfected cells. Garry, R. F., AIDS 3:683-694 (1989). This may explainresults obtained when the inventors used the North American isolatenumber 1 (N. Amer. #1, Table B-5). When this virus was used to infectPBMC's, in the absence of drug, a CD4/CD8 ratio of 0.35 was observed 7days post-infection. At the same time analysis of the culture mediumfrom cells infected with this isolate revealed the presence of viral p24antigen (Table B-4) which suggested that a productive viral infectionhad occurred.

Time of drug addition studies

T30177, DS5000 or AZT was added to MT-4 cells infected with HIV-1_(IIIB)(MOI of 1) at various times post-infection. Test compounds were added ata concentration 100-fold higher than the determined IC₅₀ value for eachdrug in the standard assay performed using MT-4 cells and the IIIBstrain of HIV-1 (Table B-2). Viral p24 antigen levels were monitored 29hour post-infection. The results of this assay indicate that postponingthe addition of T30177 for one hour was enough to dramatically reducethe inhibitory effects of this compound in a fashion similar to that ofDS5000 and clearly different from AZT which lost its protective capacitywhen added to the cell culture medium 3 or 4 hours post-infection (FIG.18). A similar result was obtained when comparing T30177 with CSB, aknown inhibitor of both virus binding to cells and fusion related events(Clanton, et al., J. Aids 5:771-781 (1992)), in that the antiviralactivity of both T30177 and CSB was greatly reduced if added to infectedcell cultures one hour post-virus infection (data not shown).

HeLa-CD4β-galactosidase cell studies

To differentiate the effects of T30177 on early events in the viral lifecycle, through integration and subsequent production of the tat geneproduct, from the inhibition of HIV-1 gp 120-mediated cell fusion twoexperimental protocols were employed. The first protocol monitored theeffects of the drug on the ability of HIV-1_(RF) to infect and/orreplicate within HeLa-CD4-LTR-β-galactosidase cells and was performed asdescribed in Methods. In this experiment drug interdiction at any stepin the viral life cycle through the production of the tat gene productwould cause a decrease in expression of the β-galactosidase gene, thetranscription of which is regulated by the HIV-1 LTR. The results showthat T30177 is a potent inhibitor of β-galactosidase production in thisassay with an IC₅₀ value of 0.009 μM, while the IC₅₀ value obtained forCSB in the same experiment was 0.26 μM (FIG. 19A). In controlexperiments T30177 had no observable direct effect on β-galactosidaseenzyme activity at concentrations up to 10 μM (data not shown).

The second protocol used was a virus-free assay designed to monitor CD4-and gp120-mediated cell fusion events. In this assay T30177 was able tointerfere with the fusion process (FIG. 19B). However, the observed IC₅₀value (1 μM) was approximately 100-fold higher than that needed tointerfere with β-galactosidase production in the virus infection assay(FIG. 19A). In the same assay system the IC₅₀ value observed for CSBincreased approximately 3-fold to 0.8 μM over the concentration neededto interrupt β-galactosidase production in the virus infection assay(FIG. 19).

The three-dimensional structure of an oligonucleotide with the sequenceof T30177 is stabilized by the formation of an intramolecular G-octet,(Rando, et al, J. Biol. Chem. 270:1754-1760 (1995)). Previously theinventors have reported how the replacement of one of the Gs involvedwith tetrad formation with a deoxyadenosine (A) reduced the anti-HIV-1activity of the resultant molecule. Rando, et al, J. Biol Chem.270:1754-1760 (1995). To determine the effects of intramolecular tetradformation in T30177 on the observed inhibition of β-galactosidaseproduction in the two assays presented in FIG. 19, the inventors testedT30526, an oligonucleotide in which a dA has been substituted for a dGat a position that would interrupt the formation of one of the twotetrads involved in the G-octet. T30526 has the same partial PT patternsas T30177 (Table B-7). T30526 has the same partial PT pattern as T30177(Table B-7). T30526 was found to be approximately 100-fold less potentthat T30177 in inhibiting HIV-1_(RF) production in culture assays (TableB-7), 10-15-fold less potent at inhibiting virus-infected cellβ-galactosidase production (FIG. 19A) and did not inhibit cell fusion atthe highest concentration of drug tested (20 μM, FIG. 19B).

TABLE B-7 Inhibition of various HIV-1 strains in culture assays and theHIV-1 integrase enzyme in vitro. Antiviral Assay^(a) Anti-IntegraseAssay^(b) IC₅₀(μM) IC₅₀μM) Compound nucleotide sequence backbone^(c) RFSKI IIIB 3′-proc strand tran. G-0ctet anti-HIV-1: T301755′-GTGGTGGGTGGGTGGGT-3′ PD 6.58 — — 0.170 0.125 T301775′-GTGGTGGGTGGGTGGGT-3′ pPT 0.075 0.025 2.83 0.092 0.046 T300385′-GTGGTGGGTGGGTGGGT-3′ PT 0.030 — — 0.090 0.070 T305265′-GTGATGGGTGGGTGGGT-3′ pPT 11.7 — — 0.200 0.123 G-0ctet thrombinbinding: T30340 5′-GGTTGGTGTGGTTGG-3′ PD > — — >0.50 >0.5 T306595′-GGTTGGTGTGGTTGG-3′ pPT 100.0 2.81. >20.0 >0.50 >0.5 T303415′-GGTTGGTGTGGTTGG-3′ PT >20.0 — — 0.042 0.023 4.76 Antisense,Anti-HIV-1: T30658 5′-TCTTCCTCTCTCTACCCACGCTCIC-3′PD >20.0 >20.0 >20.0 >0.5 >0.5 T30662 pPT >20.0 >20.0 >20.0 >0.5 >0.5T30531 5′-TCTTCCTCTCTCTACCCACGCTCIC-3′ PT 0.17 — — 0.030 0.0365′-TCTTCCTCTCTCTACCCACGCTCIC-3′ Control Compounds: 0.007 0.0220.002 >1.0 >1.0 AZT 0.016 — 0.031 0.07 0.06 DS500^(d) 0.057 0.260.078 >1.0 >1.0 ddC 0.02 0.09 0.09 — — UC38 1.7 1.6 0.6 — — CSB^(a)Antiviral assay results were obtained from infection of CEM-SS orMT4 cells with the indicated virus strain. The results are the averagesof three or more experiments. ^(b)Anti-integrase results were obtainedfrom experiments designed to monitor the 3′-processing orstrand-transfer activities of the enzyme, the results presented are theaverages of three or more experiments. ^(c)Oligonucleotides weresynthesized with either total phosphodiester (PD) backbone, totalphosphorothioate (PT) backbone, or partial phosphorothioate (pPT)backbone, in which the 5′-and 3′-penultimate internucleoside linkageswere phosphorothioate. ^(d)For DS5000 the μM units are an approximationbased upon the average molecular weight (5000) of the material used inthese studies.

Long term suppression of HIV-1

In separate experiments, HIV-1_(IIIB) infected MT-4 cells were treatedwith T30177, AZT, DS5000, or the bicyclam compounds JM2763 or JM3100 forfour days using drug concentrations equivalent to 1, 10 or 100-fold overtheir respective IC₅₀ values (Table B-7). The IC₅₀ values used forJM2763 and JM3100 were from previously reported results, (De Clereq, etal., Antimicrob. Agents Chemother. 38:668-674 (1994)). After four daysin culture the cells were washed and then further cultured in completemedium without drug. The cells were monitored daily for the appearanceof viral-induced syncytium formation and every second or third day forviral p24 antigen in the culture medium. In cells treated with T30177,at 100-fold over the IC₅₀ value (approximately 10 μM), suppression ofvirus P24 production was observed for at 1st 27 days after removal ofdrug from the infected cell culture (FIG. 20). Furthermore, there was nodetectable viral cDNA (by PCR analysis) in cells examined up to 11 daysafter the removal of T30177 from the infected cell culture (data notshown). Cells treated in the same fashion with AZT, DS5000, JM2763, orJM31000 had measurable levels of viral p24 antigen in the culture mediumwithin 3 days after removal of the drug (FIG. 20). The degree ofcontinued suppression was contingent upon the concentration of T30177used in the assay and the duration of the drug treatment regimen (datanot shown). The concentration and duration of treatment regimen data areconsistent with those previously reported for 1100-15, (Rando, et al, J.Biol. Chem. 270:1754-1760 (1995)).

To determine if exposure of cells to T30177 protects them for subsequentinfection with HIV-1, cultures of HIV-1 infected MT-4 cells treated for4 days with T30177 (100-fold over the IC₅₀ value) were washed and thenreinfected with HIV-1_(IIIB) before resuspension in fresh culture mediumwithout drug. In these assays there was no protection of cells from thesecond round of viral infection (data not shown).

Single cycle analysis of viral cDNA

Total DNA from HIV-1_(SKI) infected CEM-SS cells was isolated 12 hourpost-infection and analyzed for the presence of viral cDNA as describedin Methods. In this experiment viral cDNA was detected in cells treatedwith 1 or 10 μM T30177 (approximately 10- to 100-fold over the IC₅₀value) even when the drug was added to the cell culture at the time ofvirus infection (FIG. 21). This is in contrast to the results obtainedwhen the adsorption blocking drug CSB (10 μM), the nucleoside RTinhibitor ddC (10 μM), or the nonnucleoside RT inhibitor UC38 (1 μM)were used as control drugs. UC38 is an analog of oxathiincarboxanilide.Bader, et al., Proc. Natl. Acad. Sci. U.S.A. 88:6740-6744 (1991);McMahon, et al., Proc. Natl. Acad. Sci. USA (1995). As expected therewas no detectable viral DNA in cells treated during, or very soon after,virus infection with any of the three control drugs when used atconcentrations 10- to 100-fold over their reported IC₅₀ values (TableB-7, FIG. 21).

Analysis of replicated viral DNA

The inventors have previously reported on the presence of viral cDNA inT30177 treated SUP T1 cells 36 hour post infection with a lower MOI(multiplicity of infection of virus) of HIV-1_(DV). Rando, et al, J.Biol. Chem. 270:1754-1760 (1995). As described above, viral cDNA wasalso detected in T30177 treated cells 12 hour post-infection with a highMOI of virus (FIG. 21). To determine the extent of viral replicationwithin these cells PCR primers were used which would differentiatebetween the different stages of viral replication through the productionof circular proviral DNA (2-LTR circles). The results of theseexperiments indicate that viral replication has occurred in the T30177treated cells up to an including the production of 2-LTR circles (FIGS.22A-D).

Inhibition of viral enzymes

Oligonucleotides with PT backbones have been reported to be much morepotent inhibitors of HIV-1 reverse transcriptase (RT) than the samemolecules with PD backbones. Ojwang, et al., J. AIDS 7:560-570 (1994).T30177 was able to inhibit HIV-1 RT however, the concentration needed toinhibit the enzyme by 50% was above 5 μM when gapped duplex DNA orRNA:DNA templates were used (Table B-8). It is interesting to note thatwhen the primed ribosomal RNA template was used the IC₅₀ value forT30177 was in the 1 μM range (Table B-8).

TABLE B-8 Inhibition of recombinant HIV-1 Reverse Transcriptase.IC₅₀(μM) Template T30177 AZT 5′-triphosphate poly(rA) · p(dT) 12-18 11.00.59 4.2 0.6 gapped duplex DNA 8.0 0.47 10.0 0.40 ribosomal RNA 1.20.019 0.36 0.008 ^(a)The concentration required to inhibit enzymeactivity by 50% (IC₅₀) is given for duplicate experiments in μM units.

T30177 was also tested for its ability to inhibit HIV-1 protease andintegrase enzymes. When concentrations of T30177 up to 10 μM were usedin protease inhibition assays no effect on the viral enzyme was observed(data not shown). However, when assayed for its effect on HIV-1integrase, T30177 was able to reduce both the 3′-processing and strandtransfer activities of the integrase enzyme with IC₅₀ values of 0.092and 0.046 μM, respectively (Table B-7).

To determine if the sequence, three dimensional structure, chemicalcomposition of the backbone or a combination of these parameterscontributed to the observed anti-integrase activity of T30177, theinventors synthesized and tested for enzyme inhibitory activity theoligonucleotides shown in Table B-7. T30038, T30175, and T30526 arevariations of T30177. T30340, T30341 and T30659 are variations of thethrombin-binding aptamer sequence reported by Bock et al. Bock, et al.,Nature 355:564-566 (1992). Both the dG-rich sequence of the anti-HIV-1oligonucleotide T30177 and the thrombin binding aptamer have been shownto fold upon themselves to form structures stabilized by intramolecularG-octets. Rando, et al, J. Biol. Chem. 270:1754-1760 (1995).; Schultze,et al., J. Mol. Biol. 235:1532-1547 (1994); Wang, et al., Biochem.32:1899-1904 (1993). Oligonucleotides T30531, T30658 and T30662 arevariations of the antisense compound GEM91 reported to be a potentinhibitor of HIV-1. Agrawal et al., Antisense Research and Development2:261-266 (1992).

The IC₅₀ values for each of these oligonucleotides tested in theintegrase assay are shown in Table B-7. The results of this experimentindicate that any of the sequence motifs tested were potent inhibitorsof the HIV-1 integrase enzyme when the oligonucleotides were synthesizedwith a PT backbone. When the number of PT linkages in the backbone wasreduced to one linkage at each end of the molecule (pPT) the thrombinbinding aptamer (T30559) and the antisense sequence (T30662) no longerdisplayed anti-integrase activity while the level of inhibition observedusing T30177 was relatively the same as that observed using the total PTversion of this molecule (T30038). For compounds with total PD backbonesonly the total PD version of T30177 sequence motif was able to inhibitviral integrase with IC₅₀ values of 170 and 125 nM for the 3′ processingand strand transfer enzyme activities, respectively. T30526, thetetrad-disrupted mutant version of T30177, was still able to inhibitviral integrase protein in this assay, albeit at a concentration 2- to3-fold higher than that observed using T30177.

Conclusions of the In Vitro HIV Inhibition Studies

The inventors expanded upon the earlier observations of their initialstudies (see also, Ojwang, et al., J. AIDS 7:560-570 (1994); Rando, etal, J. Biol. Chem. 270:1754-1760 (1995)) on the anti-HIV-1 activity ofdG-rich oligonucleotides by demonstrating the efficacy of T30177 againstmultiple laboratory strains and clinical isolates of HIV-1. Using thecytotoxicity (Table B-1) and the efficacy data (Tables B-2 and B-4), itwas found T30177 to have a wide range of therapeutic indices TIs)depending upon the viral strain and cell line used in a given assay. Forexample, when T30177 was used to inhibit HIV-1_(SKI) in CEM-SS cells aTI of 3680 was obtained. However, when measuring the effect onHIV-1_(RF) in MT2 cells, the TI for T30177 was only 226.

The variability in efficacy of T30177 in PBMCs and PBLs, which dependedupon the clinical isolate tested, was very similar to the variation inactivity observed for the nucleoside analogs AZT and ddI. It isinteresting to note that an approximately 20 fold variation in the IC₅₀value was observed for T30177 when used to inhibit HIV-1_(IIIB) inCEM-SS cells (2.8 μM) versus PBMSc (0.12 μM) (Tables B-2 and B-3). Anexplanation for this observation may be that when viruses are propagatedcontinuously in homogeneous cell lines the “adapt” to those cells andbegin to display phenotypes different from low passage clinicalisolates. Therefore, results obtained using clinical isolates to infectheterogeneous populations of primary cells (PBMCs or PBLs) may be morepredictive of in vivo efficacy than data generated using laboratorystrains of HIV-1 in established cell lines. It is unlikely thatHIV-1_(IIIB) is a resistant strain of HIV-1 since T30177 was moreeffective against this virus in PBMCs than in cell lines. However, giventhe well documented ability of HIV-1 to mutate and thus developresistance to known therapies, efforts are underway to determine ifresistant mutants can arise after treatment of HIV-1 infected cells withT30177.

In mechanism of action studies it was found that T30177 displayed someantiviral activity which indicated a mechanism of action similar to theknown blockers of virus adsorption or virus mediated cell fusion such asdextran sulfate and CSB (FIGS. 18 and 19). Like CSB and DS5000, T30177needed to be added to cells at the time of or soon after virusinfection. However, T30177 is 100-fold less effective in inhibiting gp120-induced cell fusion events than it is at inhibiting early events inthe viral life cycle, suggesting a specific point of interdiction withvirus distinct at least from that of CSB. In addition, the antiviralprofile of T30177 also displayed other characteristics whichdistinguished T30177 from DS5000 and CSB. For example, while DS5000 isactive against a wide range of enveloped viruses, T30177 appears to be amore selective inhibitor of retroviruses with maximum efficacy displayedwhen used to inhibit strains of HIV-1 (Tables B-2 and B-3).

Experimental results presented in FIG. 21 show that unlike control drugsCSB, AZT, and UC38, when T30177 was added to cell cultures during virusinfection it was unable to completely block viral infection even whenused at concentrations 100-fold over the IC₅₀ value (˜10 μM).Furthermore, analysis of viral DNA demonstrated that viral replicativeintermediates including circular proviral DNA were present in infectedcells treated with T30177 (FIGS. 21 and 22). This data, coupled with theability of T30177 to completely suppress virus outbreak (FIG. 20), andpossibly clear virus from infected cell cultures, after removal of drugfrom infected cells (a profile not observed for AZT, DS5000, JM2763 orJM3100), suggests that a second mechanism of action, distinct frominhibition of virus binding or inhibition of cell fusion events, is atwork. One possible alternative mechanism is that T30177 interferes withthe viral integration process. A combination of activities includinginhibition of virus attachment or internalization, virus-mediated cellfusion events and viral integration could explain the loss of virus frominfected cell cultures. This typothesis is supported by the observationthat T30177 is a potent inhibitor of HIV-1 integrase function in vitro(Table B-7) and by the observed accumulation of circularized proviralDNA in the low-molecular weight Hirt DNA fractions (FIG. 22).

It is clear that highly charged molecules such as DS5000 andoligonucleotides with total PT backbones are excellent inhibitors of theintegrase enzyme in vitro. However, since T30177, of all the pPTmolecules tested maintained its level of enzyme inhibitory activity(Table B-7), it is unlikely that the mechanism of inhibition is totallybased upon a polyanion effect as seen for compounds such as DS5000 andsuramin. Carteau, et al., Arch. Biochem. Biophys. 305:606-610 (1993). Itis unclear at this time whether the G-octet structure, with the two baselong dG loops, found in T30177 is of paramount importance for inhibitionof viral integrase since the G-octet sequence found in T30659 did notinhibit integrase activity while T30526 (tetrad disrupting mutant) wasable to inhibit enzyme activity albeit at a reduced level.

While the time of drug addition studies would suggest interference withvirus internalization as a key mechanism of action for T30177 it is alsoclear that readily detectable viral nucleic acids do enter the cells. Itis quite possible that T30177 inhibits HIV-1 via several differentmechanisms of action. Another possibility is that T30177 is carried intothe cell along with the infecting virus or is slow to accumulate withincells (Bishop et al. 1996 J. Biol. Chem. 271:56988-5703) hence the needto add drug during virus infection. Experiments designed to addressthese possibilities are underway.

The recently reported emergence rate of drug-resistant virus to currentapproved therapies for HIV-1 (T_(¼) of approximately 2 days) suggeststhat single drug therapy for this virus cannot succeed (Ho, et al.,Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122 (1995), andtherefore, a likely treatment regimen for any new drug candidate wouldbe in combination with one or more other drugs which have differingantiviral mechanisms of action. Further experimentation might determinethat the actual mechanism of action for T30177 may not be via eitherinhibition of virus binding/internalization or inhibition of viralintegration, however, it is unlikely that this oligonucleotide is actingvia the same mechanism as drugs currently in use for HIV-1. Inadditional studies the Applicants have determined that T30177 is stablein serum and within cells, with a half-life measured in days (Bishop, etal. J. Biol. Chem. 1996 271:5698-5703). This information taken togetherwith the ability of T30177 to suppress HIV-1 for over four weeks afteran initial treatment regimen, in culture, makes this class of compoundsan attractive candidate for development of oligonucleotide-basedtherapeutic agents for HIV-1.

C. Site of Activity Studies-Viral Integrase Inhibition

Next the inventors undertook studies to demonstrate the potentinhibition of HIV-1 integrase by oligonucleotides containingintramolecular guanosine quartets or octets abbreviated (G4s) and toprovide better understanding of the structure-activity results from aseries of these structures and the site of molecular interactions withHIV-1 integrase. The relevance of these findings with respect to HIV-1integrase binding to its DNA substrate and to dimerization of theretroviral genome was also reviewed.

Materials Used in Site of Activity Studies

Preparation of oligonucleotide substrates and inhibitors

The following HPLC purified oligonucleotides were purchased from MidlandCertified Reagent Company (Midland, Tex.): AE117,5′-ACTGCTAGAGATTTTCCACAC-3′; AE118, 5′-GTGTGGAAAATCTCTAGCAGT-3′; AE157,5′-GAAAGCGACCGCGCC-3′; AE146, 5′-GGACGCCATAGCCCCGGCGCGGTCGCTTTC-3′;AE156, 5′-GTGTGGAAAATCTCTAGCAGGGGCTATGGCGTCC-3′; AE118S,5′-GTGTGGAAAATCTCTAGCA-3′; RM22M, 5′-TACTGCTAGAGATTTTCCACAC-3′. TheAE117, AE118, and the first 19 nucleotides of AE156, correspond to theU5 end of the HIV-1 long terminal repeat (LTR).

To analyze the extents of 3′-processing and strand transfer using 5′-endlabeled substrates, AE118 was 5′-end labeled using T₄ polynucleotidekinase (Gibco BRL) and y-[^(32P)]-ATP (Dupont-NEN). The kinase washeat-inactivated and AE117 was added to the same final concentration.The mixture was heated at 95° C., allowed to cool slowly to roomtemperature, and run on a G-25 Sephadex quick spin column (BoehringerMannheim) to separate annealed double-stranded oligonucleotide fromunincorporated label.

To analyze the extent of strand transfer using the “precleaved”substrate, AEI118S was 5′-end labeled, annealed to AE117, and columnpurified as above.

To analyze the choice of nucleophile for the 3′-processing reaction,AE118 was 3′-end labeled using α-[^(32P)]-crdycepin triphosphate(Dupont-NEN) and terminal transferase (Boehringer Manheim). Engleman, etal, Cell 67, 1211-1221 (1991); Vink, et al., Nucleic Acids Res. 19,6691-6698 (1991). The transferase was heat-inactivated and RM22M wasadded to the same final concentration. The mixture was heated at 95° C.,allowed to cool slowly to room temperature, and run on a G-25 spincolumn as before.

To determine the extent of 30 mer target strand generation duringdisintegration, Chow, et al., Science 255, 723-726 (1992), AE157 was5′-end labeled, annealed to AE156, AE146, and AE117, annealed, andcolumn purified as above.

Oligonucleotides composed of deoxyguanosine and thymidine weresynthesized, purified, and incubated with potassium ion to generate theG4s. The guanosine quartet (G4) forming structures were then purified aspreviously described. Rando, et al., J. Biol. Chem. 270, 1754-1760(1995).

Integrase proteins and assays

Purified recombinant wild-type HIV-1 integrase, deletion mutantsIN¹⁻²¹², IN⁵⁰⁻²⁸⁸, IN⁵⁰⁻²¹², Bushman, et al., Proc. Natl. Acad. Sci.U.S.A. 90, 3428-3432 (1993), and IN¹⁻⁵⁵ and site-directed mutantsINF^(F185K/C280S) and IN^(F185K/C280S/H12N/H16N) were generous gift T.Jenkins and R. Craigie, Laboratory of Molecular Biology, NIDDK, NIH,Bethesda, Md. Dr. Craigie also provided the expression system for thewild-type HIV-1 integrase. A plasmid encoding the HIV-2 integrase wasgenerously provided by Dr. R. H. A. Plasterk (Netherlands CancerInstitutes). Purified recombinant wild-type FIV and SIV integrases weregenerous gifts of Drs. S. Chow (UCLA) and R. Craigie (NIDDK),respectively.

Integrase was preincubated at a final concentration of 200 (for HIV-1and HIV-2) or 600 nM (for FIV and SIV) with inhibitor in reaction buffer(50 mM Nacl, 1 mM HEPES, pH 7.5, 50 μM dithiothreitol, 10% glycerol(wt/vol), 7.5 mM MnCl₂ or MgCL₂ (when specified), 0.1 mg/mL bovine serumalbumin, 20 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mMMOPS, pH 7.2) at 30° C. for 30 minutes. When magnesium was used as thedivalent metal ion, polyethylene glycol was added at a finalconcentration of 5% to increase activity as previously described(Engelman & Craigie, 1995). Preincubation for 30 minutes of the enzymewith inhibitor was performed to optimize increases the inhibitoryactivity in the 3′-processing reaction (Fesen et al., 1994). Then, 30 nMof the 5′-end ³²P-labeled linear oligonucleotide substrate was added,and incubation was continued for an additional 1 hr. The final reactionvolume was 16 μL.

Disintegration reactions, Chow, et al., Science 255, 723-726 (1992),were performed as above with a Y oligonucleotide (i.e., the branchedsubstrate in which the U5 end was “integratred” into target DNA) wasused.

Electrophoresis and quantitation

Reactions were quenched by the addition of an equal volume (18 μL) ofloading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol,0.025% brornophenol blue). An aliquot (5 μL) was electrophoresed on adenaturing 20% polyacrylamide gel (0.09 M Tris-borate pH 8.3, 2 mM EDTA,20% acrylamide, 8M urea). Gels were dried, exposed in a MolecularDynamics Phosphorimager cassette, and analyzed using a MolecularDynamics phosphorimager (Sunnyvale, Calif.). Percent inhibition wascalculated using the following equation:

100×[1−(D−C)/(N−C)],

where C, N, and D are the fractions of 21 mer substrate converted to l9mer (3′-processing product) or strand transfer products for DNA alone,DNA plus integrase, and integrase plus drug, respectively. IC₅₀ wasdetermined by plotting the drug concentration versus percent inhibitionand determining the concentration which produced 50% inhibition.

UV crosslinking experiments

The method used has been described by Engleman et al. Engelman, et al.,J. Virol. 68, 5911-5917 (1994). Briefly, integrase (at the indicatedconcentration) was incubated with substrate in reaction buffer as abovefor 5 minutes at 30° C. Reactions were then irradiated with a UVtransilluminator (254 mm wavelength) from 3 cm above (2.4 mW/cm₂) atroom temperature for 10 minutes. An equal volume (16 μL) of 2×SDS-PAGEbuffer (100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2%bromophenol blue, 20% glycerol) was added to each reaction. Twenty μLaliquots were heated at 95° C. for 3 minutes prior to loading on a 12%or 18% SDS-polyacrylamnide gel. The gel was run at 120 V for 1.5 hours,dried, and exposed in a Phosphorimager cassette. For inhibition of DNAbinding experiments (FIG. 21), integrase (200 nM) was preincubated withthe guanosine quartet (at the indicated concentration) for 30 minutes at30° C. prior to the subsequent addition of the radiolabeled viral DNAsubstrate (20 nM). For the competition experiments (FIG. 29), integrase(200 nM) was preincubated with either the radiolabeled viral DNAsubstrate (20 nM) or T30177 (20 nM) for 5 minutes at 30° C. prior to theaddition of competitor DNA at the indicated concentration.

Results of the Site of Activity Studies

Guanosine quartet oligonucleotides inhibit HIV-1 integrase

The inhibition of HIV-1 integrase by a series of oligonucleotides whichcan form G4s is shown in FIG. 23. Oligonucleotides T30177 and T30659 (avariant of the thrombin binding optimer shown in Table C-1)(Ojwang, etal., Antimicrob. Agents Chemother. 39, 2426-2435 (1995)) fold uponthemselves into structures stabilized by two G4s stacked upon each otherto form a guanosine octet (Rando, et al., J. Biol. Chem. 270, 1754-1760(1995); Schultze, et al., J. Mol. Biol. 235, 1532-1547 (1994)).Interestingly, T30177 (SEQ ID NO.87) is active against HIV-1 in cellculture and against purified HIV-1 integrase in vitro (Ojwang, et al.,Antimicrob. Agents Chemother. 39, 2426-2435 (1995)) while T30659 (SEQ IDNO.53) is not. For example, inhibition of both the 3′-processing andstrand transfer activities of HIV-1 integrase (FIGS. 23A) by T30177 wasobserved in the nanomolar range (see FIG. 23B).

In order to ascertain why T30177 was effective and T30659 was not, theinventors made a series of compounds to incrementally change onecompound into the other. The structures of these compounds are shown inpanels C and D of FIG. 23. The differences between T30177 and T30659(i.e., the presence of additional bases at both ends, differentsequences in all three loops, and extension of loop 2) manifestthemselves in dramatic increases in the IC50 values (FIG. 23D). Todistinguish the contributions of each of these changes, the inventorsfirst added the same 5′- and 3′-nucleotides to T30659 as are present onT30177, yielding T30674 (a variant of SEQ. ID. NO. 33 shown in TableC-1) (FIG. 23C). These changes did not confer potency (FIG. 23D). Thenit was undertaken to change either loop 1 to obtain T30675 (FIG. 23C) orthe three bases in loop 2 into those found in T30177, yielding compoundT30677 (FIG. 23C). Neither change by itself conferred potency (FIG.23D). However, when the change was accomplished in two of the loops toresemble T30177, yielding T30676 or T30678 (variants of SEQ. ID. NO. 33shown in Table C-1) (FIG. 23C), the inventors were able to significantlyimprove the activity over that of T30659. Interestingly, a two- tothree-fold decrease in potency was also observed when a second quartetwas unable to form, yielding T30526 (FIG. 23D). These data suggest notonly that the octet structure is critical but also that the loops areimportant for interaction with HIV-1 integrase.

The activities of the oligonucleotides in the cellular assays did notstrictly correlate with the in vitro anti-integrase activity (FIG. 23D).The correlation is complicated by the differential stabilities andsusceptibilities to nuclease digestion of the oligonucleotides in vivo(Joshua O. Ojwang and Robert F. Rando, unpublished).

In FIG. 23, G4 oligonucleotides were initially tested in a dual assaywhich measures both 3′-processing and strand transfer. Craigie, et al.,Cell 62, 829-837 (1990); Katz, et al., Cell 63, 87-95 (1990). A strandtransfer assay using “preprocessed” (3′-recessed) substrate (19 mer inFIG. 24A, left panel) was also performed to determine whether the strandtransfer reaction was truly being inhibited or whether the inhibition ofthe 3′-processing reaction caused the decrease in the subsequent strandtransfer products. Inhibition of strand transfer using this substratewas observed in the same concentration range (FIG. 24A, right panel) asthat seen with the blunt-ended, duplex oligonucleotide substrate (FIG.23A, top). Therefore, G4 oligonucleotides inhibit both steps of theintegrase reactions: 3′-processing and strand transfer.

Inhibition of 3′-processing was confirmed using DNA substrates labeledat the 3′-end, (Engleman, et al, Cell 67, 1211-1221 (1991); Vink, etal., Nucleic Acids Res. 19, 6691-6698 (1991)) (FIG. 24B, left panel),showed that all of the G4s tested inhibited glycerolysis, hydrolysis,and circular nucleotide formation to the same extent (FIG. 24B, rightpanel). Thus, G4 oligonucleotides exert a global inhibition of the threenucleophiles in the 3′-processing reaction (glycerol, water, or thehydroxyl group of the viral DNA terminus).

Having demonstrated that the catalytic activities of integrase could beinhibited by G4 oligonucleotides, the inventors next examined whetherDNA binding was also affected, They performed UV crosslinking ofintegrase-DNA reactions to address this question. Crosslinking ofsubstrate DNA to integrase followed by electrophoresis results in aproduct having a molecular weight of approximately 39 kDa (Engleman etal., 1994, Yoshinaga et al., 1994). As seen in FIGS. C-3A and C-3B,binding of HIV-1 integrase to radiolabeled U5 DNA substrate wasinhibited by preincubation of the enzyme with T30177 in the sameconcentration range as its IC₅₀ value for strand transfer (lanes 3-7).In contrast, preincubation of the enzyme with T30659, which was poorlyactive in the 3′-processing/strand transfer assay (FIG. 23D), resultedin only modest inhibition of DNA binding even at a T30659 concentrationof 500 nM (FIG. 25A, lanes 9-13).

Importance of the HIV-1 integrase zinc finger region for guanosinequartet oligonucleotide interactions

Integrase can catalyze in vitro an apparent reversal of the DNA strandtransfer reaction called disintegration. Chow, et al., Science 255,723-726 (1992). In contrast to the 3′-processing and strand transferreactions, disintegration requires neither the N-terminal zinc-fingerregion nor the C-terminal DNS-binding domain of integrase. Bushman, etal., Proc. Natl. Acad. Sci. U.S.A. 90, 3428-3432 (1993). For thisreason, the HIV-1 integrase catalytic core domain, In⁵⁰⁻²¹² (FIG. 26A),can be use din the intramolecular disintegration assay and for testingthe site of action of inhibitors. Mazumder, et al., Proc. Natl. Acad.Sci. 91, 5771-5775 (1994); Mazumder, et al., AIDS Res. Hum. Retrov. 11,115-125 (1995).

In the disintegration assay, only the In¹⁻²⁸⁸ and IN¹⁻²¹² proteins (FIG.26B) were inhibited by T30177 (with IC₅₀s of 270 and 600 nM,respectively) while neither IN⁵⁰⁻²¹² (FIG. 26B) nor IN⁵⁰⁻²⁸⁸ (data notshown) showed more than 30% inhibition at a 3 μM concentration ofT30177. The concentration of T30177 required for inhibition ofdisintegration was higher than that required for inhibition of either3′-processing or strand transfer. These results are consistent withthose observed with other molecules (Fesen et al., 1994, Mazumder et al,1994). This observation suggests that the active site of HIV-1 integrasemay tolerate drug-induced protein or DNA distortion during thedisintegration reaction, consistent with the relative tolerance ofintegrase to mutagenesis of either substrate features (Chow & Brown,1994) or protein structural domains (Bushman et al., 1993) in thisreaction. This is the first example of an HIV-1 integrase inhibitorrequiring the enzyme zinc-finger region for inhibitory activity. Theseresults suggest that the zinc-finger may assist in stabilizing bindingto T30177.

This hypothesis was investigated further by monitoring binding ofwild-type, full length integrase (IN¹⁻²⁸⁸) and of the deletion mutantsto radiolabeled T30177. The concentration of T30177 required forDNA-protein complex formation was the same as that required for complexformation using the viral U5 DNA substrate (i.e., in the 20 nM range).UV crosslinking assays, Engelman, et al., J. Virol. 68, 5911-5917(1994), showed that INI¹⁻²⁸⁸ formed a DNA-protein complex of theexpected molecular weight in the absence or presence of added manganese(FIG. 26C, lanes 8 and 9). The IN¹⁻²¹² protein, which has previouslybeen shown to bind to linear viral DNA only at high concentrations(approximately 2.56 μM) and only in the presence of divalent metal ion,(Engelman, et al., J. Virol. 68, 5911-5917 (1994)), was able tocrosslink to T30177 with the same efficiency as wild-type integrase inthe absence or presence of added manganese (lanes 2 and 3). The IN⁵⁰⁻²⁸⁸protein, which contains a nonspecific DNA-binding domain, was also ableto crosslink to T30177 with the same efficiency as wild-type integrasein the absence or presence of added manganese (lanes 4 and 5),consistent with its ability to bind to viral U5 DNA (Engelman et al.,1994). The extent of crosslinking was significantly diminished in thecase of the core mutant IN⁵⁰⁻²¹² compared to IN¹⁻²¹² in the absence orpresence of manganese (compare lanes 2 and 3 with 6 and 7, fastermigrating complex). The higher molecular weight species in lane 6,having the expected molecular weight of a dimer, has been reproduciblyobserved, but its density has not been confirmed. These data support thenotion that the N-terminus of HIV-1 integrase assist in the formation orstabilization of an HIV-1 integrase-T30177 complex, perhaps by bindingthe oligonucleotide.

DNA-binding activities of the HIV-1 integrase zinc finger domain

To further analyze the binding of the N-terminal zinc finger region toT30177 and compare these results to the viral U5 substrate, UVcrosslinking was performed with an In¹⁻⁵⁵ deletion mutant (FIG. 26A)containing only this domain. As seen in FIG. 27A-B, this mutant couldnot bind either the T30177 oligonucleotide or the viral DNA substratewhen only manganese or magnesium was present left and right panels,lanes 3 and 4). However, the IN¹⁻⁵⁵ protein could bind to both DNAs inthe presence of zinc and either manganese or magnesium (left and rightpanels, lanes 5 and 6). Significantly, the In¹⁻⁵⁵ protein was able tobind to the T30177 G4 oligonucleotide, but not the viral DNA substrate,in the presence of zinc alone (left and right panels, lanes 9 and 10).These results are in accord with the known zinc-binding ability of thisdomain. Bushman, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 3428-3432(1993); Burke, et al., J. Biol. Chem. 267, 9639-9644 (1992). But theyalso suggest that the N-terminal domain of integrase has DNA bindingcapabilities on its own. Finally, these experiments demonstratecomparable binding of the HIV-1 integrase zinc finger domain to anoligonucleotide containing in G4s than to a double-stranded, linear,viral DNA oligonucleotide when both manganese (or magnesium) and zincare present but more efficient binding to the G4 oligonucleotide undernon physiological conditions (zinc alone). The inventors also found thatthe nucleocapsid protein of HIV-1, a nucleic acid annealing proteinwhich contains two CCHC zinc fingers and which is essential fordimerization of the retroviral RNA genome, Tsuchihashi, et al., J.Virol. 68, 5863-5870 (1994), was able to bind efficiently to T30177(data not shown). The ability of zinc to confer DNA binding ability onthe IN¹⁻⁵⁵ protein was examined by replacement of this ion with othertransition metals. Consistent with spectroscopic data (Burke et al.,1992), only zinc was able to induce detectable DNA binding to the G4oligonucleotide (data not shown).

Increased potency of guanosine quartets in magnesium

In contrast to IN¹⁻⁵⁵, the extent of crosslinking (and presumablybinding) of wild-type integrase to radiolabeled guanosine quartet wasincreased in the presence of magnesium relative to manganese at severalconcentrations of the guanosine quarter (FIG. 28A). This observation ledus to examine whether the inhibitory activity of T30177 and analogscould also be enhanced by buffer containing magnesium. In order toaddress this question, the inventors tested three versions of T30177 asshown in FIG. 28B. T30175 has the same base sequence as T30177 (pPTvariant of SEQ. ID. NO. 33) but is composed entirely ofphosphorothiodiester internucleotidic linkages. The inhibition of3′-processing catalyzed by HIV-1 integrase by these guanosine quartetsis shown in FIG. 28C. Both T30175 and T30177 showed four to five-foldincreases in potency when magnesium was used as the divalent metalinstead of manganese. In contrast, T30038 showed no significant increasein potency when magnesium was used as the ion (FIG. 28D). These data arein accord with the increased stability constants formagnesium-nucleotide complexes when oxygen replaces sulfur (Pecoraro etal., 1984). The opposite is true for manganese. Therefore, the greaterinhibitory potency of T30177 in buffer containing magnesium versusmanganese may reflect a requirement for magnesium ion coordination alongthe phosphodiester backbone of T30177 in order to confer inhibitoryactivity and optimum interaction of T30177 with HIV-1 integrase. Thiscoordination can occur with more stability when either T30177 or T30175are assayed in buffer containing magnesium rather than manganese and ismanifested in a greater potency against 3′-processing.

DNA competition experiments

The relative affinity for the G4 oligonucleotide was probed byattempting to compete off the integrase bound to radiolabeled HIV-1viral U5 DNA with increasing concentrations of unlabeled T30177 (FIG.29A). The converse experiment, where binding of integrase toradiolabeled G4 oligonucleotide was carried out prior to the addition ofincreasing concentrations of unlabeled HIV-1 viral U5 DNA, was alsoperformed (FIG. 29B). In each case, a band having the apparent mobilityof an integrase-DNA complex was evident. In FIG. 29A, the viralDNA-integrase complex has a molecular weight of 38,500 while in FIG.29B, the T30177-integrase complex has a molecular weight of 37,000.Neither complex could not be competed off by either competitor DNA evenat concentrations where the competitor was in 500-fold excess (FIG. 29A,lane 6). Similar results were seen when the In¹⁻²¹² and IN⁵⁰⁻²¹²proteins were used in competition experiments (data not shown).Therefore, the stability of the G4 oligonucleotide DNA-integrase complexis comparable to that of the viral DNA-integrase complex is comparableto that of the viral DNA-integrase complex. Ellison, et al, Proc. Natl.Acad. Sci. U.S.A. 91, 7316-7320 (1994); Vink, et al., Nuc. Acids Res.22, 4103-4110 (1994).

Inhibition of related lentiviral integrases

T30177 was tested for inhibition of the related retroviral integrasesfrom HIV-2 (van Gent et al., 1992), simian immunodeficiency virus (SIV)and feline immunodeficiency virus (FIV) (Vink et al., 1994b). As seen inFIGS. 30A and 30B, T30177 inhibited 3′-processing catalyzed by HIV-1integrase in the expected concentration range (FIG. 30A, lanes 2-8;IC₅₀=55 nM). Inhibition of HIV-2 integrase (using HIV-1 DNA) was alsoobserved in the same range (lanes 9-15; IC₅₀=90 nM). However, FIVintegrase was inhibited at three-fold higher concentrations of T30177(lanes 16-22; IC₅₀=175 nM), and SIV integrase was inhibited atseven-fold higher concentrations to T30177 (lanes 23-29; IC₅₀=420 nM).Therefore, the T30177 G4 oligonucleotide displayed some selectivityamong the lentiviral integrases.

Conclusions Regarding the Site of Activity Studies

The present study demonstrates for the first time the binding of DNAguanosine quartet structures to HIV-1 integrase, and that someoligonucleotides recently shown to exhibit antiviral activity are potentHIV-1 integrase inhibitors.

Guanosine Quartet Oligonucleotides are Novel and Potent Inhibitors ofHIV-1 integrase

Oligonucleotides composed of deoxyguanosine and thymidine and formingguanosine-tetrads (G4) structures have previously been shown in inhibitHIV replication. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995);Wyatt, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Twomechanisms have been invoked. First, some oligonucleotides have beenshown to bind to the V3 loop of the envelope protein gp120 andsubsequently inhibit virus adsorption and cell fusion. Wyatt, et al.,Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Secondly,oligonucleotides such as those described in the present study alsoinhibit viral-specific transcripts, Rando, et al., J. Biol. Chem. 270,1754-1760 (1995), presumably by inhibiting viral integration. Ojwang, etal., Antimicrob. Agents Chemother. 39, 2426-2435 (1995). The presentfinding that inhibition of the HIV-1_(RF) strain in cell cultureparallels that of purified integrase in vitro in the series of G4oligonucleotides tested (FIG. 23D) further demonstrates the possibilitythat HIV-1 integrase can be targeted by some G4 oligonucleotides.

G4 oligonucleotides differ from previously published HIV-1 integraseinhibitors in several ways. (Table C-1) First, they are among the mostpotent inhibitors to date with IC50's in the nanomolar range. Theirpotency range is comparable to flavone, Fesen, et al., Biochem.Pharmacol. 48, 595-608 (1994), and tyrophostin derivatives, Mazumder, etal, Biochemistry 34, in press (1995), which, however, generally fail toshow antiviral activity. Secondly, the zinc finger domain of HIV-1integrase contributes to the inhibition by G4 oligonucleotides, astruncation mutant enzymes lacking this domain are resistant to the G4oligonucleotides. This property is unique, as all the other inhibitorsto date are active against the HIV integrase catalytic core domain.(Table C-2) Mazumder, et al., Proc. Natl. Acad. Sci. 91, 5771-5775(1994); Mazumder, et al., Mol. Pham. submitted (1995); Fesen, et al.,Biochem. Pharmacol. 48, 595-608 (1994); Mazumder, et al., Biochemistry34, in press (1995). Finally, G4 oligonucleotides form stable enzymecomplexes that cannot be displaced by excess viral DNA oligonucleotide.

TABLE C-1 HIV-1 Integrase Inhibitor Compound Design and List T301775′ gtggtgggtgggtgggt  -3′ pPT version of parent compound I100-15 17 mer(SEQ ID NO 87) T30038 5′ gtggtgggtgggtgggt  -3′ PT, Total PT versionT30177 17 mer (SEQ ID NO 87) T30340 5′  ggttggtgtggttgg  -3′ PD versionof thrombin binding aptamer 15 mer (SEQ ID NO 53) T303415′  ggttggtgtggttgg  -3′ pPT version of thrombin binding aptamer 15 mer(SEQ ID NO 53) T30659 5′  ggttggtgtggttgg  -3′ PT version of thrombinbinding aptamer 15 mer (SEQ ID NO 53) T30673 5′ gtggttggtgtggttgg  -3′pPT variant T30177 17 mer (SEQ ID NO 54) T306745′ gtggttggtgtggttggt -3′ pPT variant T30177 18 mer (SEQ ID NO 55)T30675 5′ gtggtgggtgtggttggt -3′ pPT variant T30177 18 mer (SEQ ID NO56) T30676 5′ gtggtgggtgtggtgggt -3′ pPT variant T30177 18 mer (SEQ IDNO 57) T30677 5′ gtggttggtg ggttggt -3′ pPT variant T30177 17 mer (SEQID NO 55) T30678 5′ gtggtgggtg ggttggt -3′ pPT variant T30177 17 mer(SEQ ID NO 56) T30679 5′ gtggttggtg ggtgggt -3′ pPT variant T30177 17mer (SEQ ID NO 58) T30660 5′ guggugggugggugggu  -3′ pPT version using2′0-methyl RNA* 17 mer (SEQ ID NO 59) T30661 5′ guggugggugggugggu  -3′pPT version RNA* 17 mer (SEQ ID NO 59) T30695 5′ g ggtgggtgggtgggt  -3′pPT variant remove T near 5′-end 16 mer (SEQ ID NO 60) T306965′ gtgggtggtgggtgggt  -3′ pPT variant invert loop 1 17 mer (SEQ ID NO61) T30697 5′ gtggtggggtggtgggt  -3′ pPT variant invert loop 2 17 mer(SEQ ID NO 62) T30698 5′ gtggtgggtggggtggt  -3′ pPT variant invert loop3 17 mer (SEQ ID NO 63) T30699 5′ gtgggtggtggggtggt  -3′ pPT variantinvert loops 1 and 3 17 mer (SEQ ID NO 64) T307005′ gtggTgggtgggtgggt  -3′ pPT variant T = c-5 propynyl dU 17 mer (SEQ IDNO 65) T30701 5′ gtggtgggtgggTgggt  -3′ pPT variant T = c-5 propynyl dU17 mer (SEQ ID NO 66) T30702 5′ gtggTgggtgggTgggt  -3′ pPT variant T= c-5 propynyl dU 17 mer (SEQ ID NO 67) T30177 5′ gtggtgggtgggtgggt  -3′pPT version of parent compound T100-15 17 mer (SEQ ID NO 87) T30719 5′ gggTgggtgggTgggt  -3′ pPT variant T = c-5 propynyl dU 16 mer (SEQ ID NO68) T30720 5′ g gggTggtgggTgggt  -3′ pPT variant T =c-5 propynyl dU 16mer (SEQ ID NO 69) T30721 5′ I ggIIggIIggIIggI  -3′ pPT variant I = dIwhere I = inosine 16 mer (SEQ ID NO 70) T30722 5′ gtgggTggtgggTgggt  -3′pPT variant T = c-5 propynyl dU 17 mer (SEQ ID NO 71) T300755′ gtggtgggtgggtgggt  -3′ 3′-cholesterol via triglycyl linker 17 mer(SEQ ID NO 72) T30570 5′ gtggtgggtgggBgggt  -3′ pPT version B = 5-BromodU 17 mer (SEQ ID NO 73) T30571 5′ gtggBgggBgggBgggt  -3′ pPT version B= 5-Bromo dU 17 mer (SEQ ID NO 74) T30576 5′ gtggIgggtgggtgggt  -3′ pPTversion I = 5-Iodo dU 17 mer (SEQ ID NO 75) T305775′ gtggtgggIgggtgggt  -3′ pPT version I = 5-Iodo dU 17 mer (SEQ ID NO76) T30578 5′ gtggtgggtgggIgggt  -3′ pPT version I = 5-Iodo dU 17 mer(SEQ ID NO 77) T30579 5′ gtggIgggIgggIgggt  -3′ pPT version I = 5-IododU 17 mer (SEQ ID NO 78) T30743 5′ gtggCgggtgggtgggt  -3′ pPT version C= cytosine 17 mer (SEQ ID NO 79) T30744 5′ gtggtgggCgggtgggt  -3′ pPTversion C = cytosine 17 mer (SEQ ID NO 80) T307455′ gtggtgggtgggCgggt  -3′ pPT version C = cytosine 17 mer (SEQ ID NO 81)T30746 5′ gtggCgggCgggCgggt  -3′ pPT version C = cytosine 17 mer (SEQ IDNO 82) T30748 5′ tgggaggtgggtctg  -3′ pPT New sequence - intermoleculartetrad 15 mer (SEQ ID NO 83) T30747A 5′ tgggaggtgggtctg  -3′ Allphosphodiester linkages 15 mer (SEQ ID NO 84) T30747B5′ tgggaggtgggtctg  -3′ All phosphodiester link. DMT at 3′-end 15 mer(SEQ ID NO 85) T30754 5′ gcggggctccatgggggtcg  -3′ pPT New sequence -intermolecular tetrad* 20 mer (SEQ ID NO 86) S9358335′ gcggggctccatgggggtcg  -3′ pPT see below* 20 mer (SEQ ID NO 86) Allcompounds were synthesized using commercially available reagents exceptfor T30075 which was synthesized with a cholesterol moiety at the 3′-endattached via a triglycyl linker as described in the paper by Rando etal. (J. Biol. Chem. 1995 270: 1754-1760]  and more extensively by Vu etal. [1994 Bioconjugate Chem. 5: 666-668]. The propynyl dU buildingblocks contain uracil bases in which a propynyl group has been added tothe c-5 position. The propynyl dC building blocks contain cytosine basesin which a propynyl group has been added to the c-5 position. Thesereagents are commercially available from Glen Research Reagent co. Otherunusual bases used such as dI (inosine) and iodo and bromo dU are alsocommercially available. pPT is shorthand for ODNs in which the linkagebetween the ultimate and penultimate bases has been changed fromphosphodiester to phosphorothioate. PT is shorthand to indicate that alllinkages are phosphorothioate. Compounds T30660 and T30661 use RNAsugars or 2′-0-methyl RNA sugars as indicated instead of 2′-deoxy sugarsas found in DNA. The thrombin binding aptamer sequence used was thefirst described by Bock et al. [Nature 1992 355: 564-566] and reportedto fold into an structure stabilized by an intramolecular tetrad by Wantet al. Biochemistry 1993 32: 1899-1904]. Compound T30747 A/B containonly natural phosphodiester linkages. T30747B is 3′-modified in that thedimethoxy trityl capping group was left attached after removal of theoligonucleotide from the solid support and subsequent purification.Usually this group is removed after synthesis of the molecule. CompoundS935833 has the following pattern of phosphorothioate (PT) linkageswhere * denotes the PT linkage: 5′-g*c*gggc*t*c*c*a*tggggg*t*c*g-3′.Compound T30754 has a pPT pattern in which only the 5′ and 3′ linkagesare PT: 5′-g*cggggctccatgggggtc*g-3′. For structures of certain of theolignucleotides listed, see FIGS. C-9, C-10, C-13, and C-14. Forpercentage inhibition of 3′-processing, and for inhibition of syncytiumformation, of certain of these same oligonucleotides, see FIGS. C-11 andC-12, respectively.

TABLE C-2 Anti-HIV-1 Integrase Activity in vitro and Anti-HIV-1 VirusProduction in Cell Culture Enzyme (IC 50 μM) Cell Culture Stran Trans3-′process IC50 (μM) T30177 5′ gtggtgggtgggtgggt  -3′ 0.079 0.049 0.075(SEQ ID NO 87) T30038 5′ gtggtgggtgggtgggt  -3′ 0.090 0.070 0.06 (SEQ IDNO 87) T30340 5′   ggttggtgtggttgg  -3′ >0.500 >0.500 >50.0 (SEQ ID NO53) T30341 5′   ggttggtgtggttgg  -3′ 0.042 0.023 4.76 (SEQ ID NO 53)T30659 5′   ggttggtgtggttgg  -3′ 0.870 0.750 >20.0 (SEQ ID NO 53) T306735′ gtggttggtgtggttgg  -3′ 0.790 0.600 >35.0 (SEQ ID NO 54) T306745′ gtggttggtgtggttggg -3′ 0.760 0.610 >50.0 (SEQ ID NO 55) T306755′ gtggtgggtgtggttggt -3′ 0.485 0.500 >30.0 (SEQ ID NO 56) T306765′ gtggtgggtgtggtgggt -3′ 0.148 0.134 1.0 (SEQ ID NO 57) T306775′ gtggttggtg ggttggt -3′ 0.725 0.620 >40.0 (SEQ ID NO 55) T306785′ gtggtgggtg ggttggt -3′ 0.098 0.120 3.95 (SEQ ID NO 56) T306795′ gtggttggtg ggtgggt -3′ 0.159 0.156 3.46 (SEQ ID NO 58) T306605′ guggugggugggugggu  -3′ >50.0 (SEQ ID NO 59) T306615′ guggugggugggugggu  -3′ 0.111 0.084 46.6 (SEQ ID NO 59) T30695 5′ gggtgggtgggtgggt  -3′ 0.060 0.020 0.07 (SEQ ID NO 60) T306965′ gtgggtggtgggtgggt  -3′ 0.122 0.013 (SEQ ID NO 61) T306975′ gtggtggggtggtgggt  -3′ 0.130 0.013 (SEQ ID NO 62) T306985′ gtggtgggtggggtggt  -3′ 0.150 0.016 (SEQ ID NO 63) T306995′ gtgggtggtggggtggt  -3′ 0.136 0.050 (SEQ ID NO 64) T307005′ gtggTgggtgggtgggt  -3′ 0.082 0.040 (SEQ ID NO 65) T307015′ gtggtgggtgggTgggt  -3′ 0.072 0.030 (SEQ ID NO 66) T307025′ gtggTgggtgggTgggt  -3′ 0.032 0.030 4.91 (SEQ ID NO 67) T301775′ gtggtgggtgggtgggt  -3′ 0.079 0.049 0.075 (SEQ ID NO 87) T30719 5′ gggTgggtgggTgggt  -3′ 0.055 0.055 (SEQ ID NO 68) T30720 5′ ggggTggtgggTgggt  -3′ 0.059 0.062 (SEQ ID NO 69) T30721 5′ IggIIggIIggIIggI  -3′ 0.070 0.088 (SEQ ID NO 70) T307225′ gtgggTggtgggTgggt  -3′ 0.129 0.134 (SEQ ID NO 71) T300755′ gtggtgggtgggtgggt  -3′ 0.110 0.095 0.07 (SEQ ID NO 72) T305705′ gtggtgggtgggBgggt  -3′ 0.090 0.070 (SEQ ID NO 73) T305715′ gtggBgggBgggBgggt  -3′ 0.055 0.050 (SEQ ID NO 74) T305765′ gtggIgggtgggtgggt  -3′ 0.065 0.050 (SEQ ID NO 75) T305775′ gtggtgggIgggtgggt  -3′ 0.065 0.055 (SEQ ID NO 76) T305785′ gtggtgggtgggIgggt  -3′ 0.060 0.050 (SEQ ID NO 77) T305795′ gtggIgggIgggIgggt  -3′ 0.060 0.055 (SEQ ID NO 78) T307445′ gtggtgggCgggtgggt  -3′ 0.105 0.060 (SEQ ID NO 80) T307455′ gtggtgggtgggCgggt   -3′ 0.100 0.100 (SEQ ID NO 81) T307465′  gtggCgggCgggCgggt   -3′ 0.150 0.150 (SEQ ID NO 82) T30747A5′ tgggaggtgggtctg      -3′ >0.250 >0.250 (SEQ ID NO 84) T30747B5′ tgggaggtgggtctg      -3′ 0.120 0.120 (SEQ ID NO 85) T307485′ tgggaggtgggtctg      -3′ 0.125 0.125 (SEQ ID NO 83) S9358335′ gcggggctccatgggggtcg -3′ 0.060 0.045 0.63 (SEQ ID NO 86) T307455′ gcggggctccatgggggtcg -3′ 0.120 0.120 (SEQ ID NO 86) The integrase andviral inhibition (cell culture) assays were performed as described byOjwang et al. [1995 Antimicrobial Agents and Chemotherapy 39:2426-2435]. Integrase inhibition assays monitor two different enzymaticactivities, the 3′-processing (3′-process) activity and the strandtransfer (stran trans) activity of the enzyme. The units for the enzymeassays are in nM while the units for the cell culture inhibition assaysare in μM.

Role of the HIV-1 Integrase Zinc Finger Region

Mutation and deletion analyses show that the zinc finger motif (H-H-C-C)of retroviral integrases is required for integration (3′-processing andstrand transfer) activity. Engelman, et al., J. Virol. 66, 6361-6369(1992). However, the structural role of this region has not beenelucidated. It has been postulated too provide DNA sequence-specificity,Bushman, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 3428-3432 (1993),stabilize DNA-enzyme, Vink, et al., Nuc. Acids Res. 22, 4103-4110(1994), and enzyme multimer complexes. Ellison, et al, J. Biol. Chem.270, 3320-3326 (1995). These data provide the first direct evidence thatthe HIV-1 integrase N-terminus region (amino acids 1-55 [FIG. 26A]) caninteract directly with viral DNA in the presence of both zinc andmagnesium (or manganese). The fact that the IN¹⁻⁵⁵ protein binds to theG4 oligonucleotides in the presence but not in the absence of zinc isconsistent with the formation of a zinc finger in this region. Burke, etal., J. Biol. Chem. 267, 9639-9644 (1992). Hence, it is possible thatthe zinc finger region can selectively bind to non-B DNA structures. Itis noteworthy that the recently solved structure of HIV-1 integrase,Dyda, et al., Science 266, 1981-1984 (1994), resembles that of the Rev CHolliday junction-resolving enzyme, Ariyoshi, et al, Cell 78, 1063-1072(1994), and of the bacteriophage Mu transposes core. Rice, et al., Cell82, 209-220 (1995). These structurally related proteins also bindmultiple double helices, generating X structure intermediates. (Forreview See Katz et al., Ann. Rev. Biochem 63:133-173 (1994) and Vink etal; Trends in Genetics 9:433-438 (1993).

Although the zinc finger region of integrase is required for inhibitionby the G4s, the IN⁵⁰⁻²¹² protein, which contains only the centralcatalytic domain, was capable of binding to T30177 (FIG. 26C). Theinventors also found that an HIV-1 integrase mutant with the two zincfinger histidines mutated to asparagines was able to bind to G4oligonucleotides (data not shown). These data suggest that HIV-1integrase may have two separate binding sites, one for viral DNA and onefor target or “host” DNA. This scenarios would be expected if integrasewere to bind both the viral and host DNA at sites which were instinctbut in close proximity in vivo. Vincent, et al., J. Virol. 67, 425-437(1993). It should be noted that the existence of a single binding siteon HIV integrase for both viral and target DNA has been proposed byothers. Vink, et al., Nucleic Acids Res. 21, 1419-1425 (1993).

Biological relevance of G4 structures

Several similarities exist between retroviral genomes and telomericregions of eukaryotic chromosomes. The two RNA strands comprising theHIV-1 genome can potentially dimerize and form intermolecular G4s invitro, Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397(1993); Awang, et al., Biochemistry 32, 11453-11457 (1993), as doestelomeric DNA. Sundquist, et al., Nature 342, 825-829 (1989). Inaddition, the β subunit of the Oxytrichia telomere binding protein hasbene proposed as a molecular chaperone for the formation of G4s at theend of chromosomes by enhancing the rate of a thermodynamically favoredtransition. Fang, et al., Cell 74, 875-885 (1993). In retroviruses, thenucleocapsid protein may also act as a molecular chaperone to enhancedimer formation. Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90,3393-3397 (1993). In this manner, it may facilitate the formation of andbind to the G4. The inventors also found that G4s can bind to purifiednucleocapsid protein (data not shown). Thus, G4s may be structurallyimportant as molecular scaffolds in both retroviral preintegrationcomplexes and telomeres, and these structures may have associatedchaperones in both cases. Finally, a G4 containing structure may act asa negative regulator of telomere elongation in vivo due to its abilityto inhibit telomerase in vitro. Zahler, et al., Nature 350, 718-720(1991). Analogously, G4 structures may act to inhibit integrase (FIG.23A-D) and thereby act as a block to auto integration or digestion ofthe viral DNA prior to insertion into the host chromosome.

The existence of G4s in vivo has not been demonstrated. However, theyhave been shown to form in vitro in telomeric sequences, Sundquist, etal., Nature 342, 825-829 (1989); Smith, et al., Nature 356, 164-168(1992); Kang, et al., Nature 356, 126-131 (1992), HIV-1 RNA sequences,Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397 (1993);Awang, et al., Biochemistry 32, 11453-11457 (1993), fragile X syndromenucleotide repeats, Fry, et al., Proc. Natl. Acad. Sci. U.S.A. 91,4950-4954 (1994), the retinoblastoma susceptibility gene, Murchie, etal., Nuc. Acids Res. 20, 49-53 (1992), immunoglobulin switch regionsequences, Sen, D., et al., Nature 334, 364-366 (1988), and possiblyduring meiotic recombination. Liu, et al., Cell 77, 1083-1092 (1994).Given these results, it is not surprising that proteins such asthrombin, Bock, et al., Nature 355, 564-566 (1992), (not normally knownto bind nucleic acids), chick topoisomerase II, Chung, et al., NucleicAcids Res. 20, 1973-1977 (1992), MyoD (a transcription factor thatregulates myogenesis), Walsh, et al., J. Biol. Chem. 267, 13714-13718(1992), an hepatocyte chromatin protein, Weisman-Shomer, et al., J.Biol. Chem. 268, 3306-3312 (1993), macrophage scavenger receptors,Pearson, et al., J. Biol. Chem. 268, 3546-2554 (1993), and a proteinfrom Tetrahymena thennophila, Schierer, et al., Biochemistry 33,2240-2246 (1994), have been found to bind G4 containing nucleic acids.Another G4 binding protein, KEM1, has been isolated and implicated inrecombination-type reactions in vivo. Liu, et al., Cell 77, 1083-1092(1994). The catalytic activities of this protein and of the integraseprotein are DNA endonucleolytic cleavage and strand transfer. However,unlike KEM1, integrase does not catalyze endonucleolytic cleavagereactions on G4s (data not shown). Thus, G4s may be mechanisticallyrelevant in a diverse set of biological processes involving enzymes withsimilar activities.

In summary, the inventors demonstrated that oligonucleotides containingintramolecular G4s are potent inhibitors of HIV-1 integrase. Inhibitionis dependent on the zinc finger region of integrase and on the structureand sequence of the G4s. These findings also suggest that novel AIDStherapies could be based upon G4s as inhibitors of HIV-1 integrase.

D. Structure-Function Studies

As shown previously, the inventors obtained evidence for inhibition ofHIV-1 infection by treatment with phosphodiester oligonucleotidescontaining only G and T bases. Additional studies noted above suggestedto the inventors that such oligomers were potent inhibitors of HIV-1integrase, in vitro. The highest activity was obtained using the 17 mer,referred to as T30177 (pPT variant of SEQ. ID. NO. 33), with compositionG12-T5. NMR evidence suggested to the inventors that T30177 forms anintramolecular fold which is stabilized by a pair of G-tetrads,connected by three single stranded loops, with a 1-2 base tail to etherside of the fold.

Thus, the inventors undertook studies to determine sequence dependenceof the intramolecular folding mechanism, in a set of four closelyrelated 16-17 base oligonucleotide homologues, with sequences in therange G10-12-T4-7. The original T30177 compound was included, along withthree derivatives which were designed so as to alter the structure ofloop domains, while keeping the pair of G-tetrads intact. Based onthermal denaturation, CD and kinetic analysis, the inventors were ableto show that a single base alteration within the loop or tail domainscan produce a very large change in folding stability. The K⁺ iondependence of these data suggested a preliminary model wherein the loopand tail domains interact to form stable metal ion-binding sites. A 16mer derivative (T30695) was designed within the context of that model,with the intent of enhancing the interaction between K⁺ and the 5′terminus of the oligomer. The inventors showed that T30695 (a variant ofSEQ. ID. NO. 33, shown in Table C-1) folding is indeed more stable thanother members of the group and is highly specific for K⁺, as assessedfrom the ion dependence of thermal denaturation, CD spectra and UVdetected folding kinetics.

To assess the relationship between biological activity and formation ofthe ion-selective oligomer fold, the inventors compared tertiarystructure stability at three K⁺ concentrations with the capacity of thefolded oligomers to inhibit the HIV-1 integrase enzyme in vitro, orHIV-1 infection in cell culture. The stability and activity data arefound to be highly correlated, as a function of sequence alteration,suggesting that formation of the stable intramolecular fold may be aprerequisite for both integrase inhibition and anti-HIV-1 activity.Although the structure of the folded state has not yet been confirmed athigh resolution, the data presented here suggested that the structure ofthe T30695 complex with K⁺ ion may be of pharmaceutical significance andcould serve as the basis for additional improvement of the observedHIV-1 activity.

Materials and Methods for the Structure/Function Studies

Oligonucleotide Synthesis

All oligonucleotides used in this study were synthesized on an AppliedBiosystems Inc. DNA synthesizer, model 380B or 394, using standardphosphoramidite chemistry, or fast deblocking Expedite chemistry on aMilligen synthesizer. All oligomers possessed 2 phosphorothioatelinkages (one on each terminus) which were introduced by theH-phosphonate method. Oligonucleotides were purified by preparativeanion exchange HPLC, on Q-Sepharose. Chain purity was confirmed byanalytical Q-sepbarose chromatography, and by denaturing electrophoresisof 32P labeled oligomers on a 20% polyaerylamide (19:1), 7M urea gelmatrix (Rando et al., (1994) J. Biol. Chem. 270, 1754-1760, 17). In allinstances, greater than 90% of the purified oligomer was determined tobe full length. Oligomer folding was monitored by native gelelectrophoresis on 15% acrylamide (19:1) matrix in TBE. Folded, 32Plabeled samples were loaded subsequent to annealing in 20 mM Li3P04, pH7.0, 10 mM KCl at 7 uM in strands, as described below.

Annealing

Prior to UV, CD or kinetic analysis, oligonucleotides were annealed at20 mM Li3P04, pH 7 at 3-15 uM in strands. Samples were heated to 90° C.for 5 min and then incubated for 1 hour at 37° C. Metal ion could beadded as the chloride either before or after the 37 ° C. incubation,with no measurable difference in final state, as assessed by UV, CD orgel analysis. As assessed by native gel electrophoresis (not shown),this annealing method was found to produce a single product withmobility consistent with a folded monomer over the strand concentrationrange from 3-15 uM, at all ion concentrations described.

Ultraviolet Spectroscopy

UV measurements were obtained on a HP 8452A diode array spectrometer,using a HP 89090A temperature regulator. Except where noted, thermaldenaturation profiles were obtained at a rate of 1.25° C./min over therange from 20° C.-80° C., on samples at 20 mM Li3P04, pH 7, at 7 uM instrands. Absorbance was monitored at 240 nm, which was determined to bethe point of maximal temperature induced change. For melting analysis,metal ion was added to the desired concentration, followed by a one hourpre-incubation at 37° C., to ensure compete annealing. Folding kineticswere obtained by manual addition of metal ion at t=0, followed byabsorption measurement at 264 nm. Mixing dead time was determined to be10 sec. Kinetics were monitored over the range from 10 sec to 15 min at25° C.

Circular Dichroism

CD spectra were obtained at 25° C. in 20 mM Li3P04, pH 7, at 15 uM instrands, on a Jasco J-500A spectropolarimeter. Metal ion was added tothe desired concentration, followed by one hour of pre-incubation at 37°C. Each spectrum in the text represents 5 averaged scans. To conform totraditional standards, data are presented in molar ellipticity(deg-cm2-dmolE-1) as measured in base, rather than strand equivalents.

Antiviral assay. The RF laboratory strain of HIV-1 was used to infectestablished cell lines for one hour at 37° C. prior to washing andresuspension in medium containing increasing concentrations of drug.Four to six days post-infection, drug treated and control wells wereanalyzed for HIV-1 induced cytopathic effects, for the presence of viralreverse transcriptase (RF) or viral p24 antigen in the culture medium aspreviously described by Ojwang et al. (Bishop et al., (1996) J. Biol.Chem. 271, 5698-5703). Purified recombinant HIV-1 integrate enzyme(wild-type) was a generous gift from Dr. Craigie, Laboratory ofMolecular Biology, NIDDK. All 3′-processing and strand-transfers wereperformed as described previously by Fresen et al. (Fresen et al. (1993)Proc. Natl. Acad. Sci. USA 90, 2399-2403) and Mazurnder et al. (Mazumderet al. (1994) Proc. Natl. Acad. Sci. USA 91, 5771-75).

Results of Structure/Function Studies

The structure of the oligonucleotides in this study are presented inFIG. 37A. For the purposes of clarity, they have been represented in thecontext of a particular folding model which places eight of theguanosincs as a central octet and the remainder of the oligomer ineither a loop region, or as part of a 1-2 base long tail region at the5′ or 3′ terminus. Previous electrophoresis and ID NMR data (Rando etal., (1994) J. Biol. Chem. 270, 1754-1760) have strongly suggested thatT30177 folds so as to form an intramolecular G-tetrad based structurewhich is stabilized by a single central G-octet. Therefore, for T30177,the simple model presented in FIG. 37A is adequately substantiated bystructural data. The validity of a similar structural model for theother members of the series is legitimately assumed based upon theirsequence similarity, to be tested in terms of the data presented below.

Thermal Denaturation Analysis

Based upon previous NMR data, and the general literature, the inventorspostulated that folding of T30177 should be strongly dependent upon K⁺binding. To quantify this, they measured the thermal stability ofT30177, as a function of added KC1 concentration. Coupled equilibriumtheory predicts that, in the instance that K⁺ binding stabilizesformation of an intramolecular fold, measured TM values should increaselinearly with the Log of the KC1 concentration. Such data are shown inFIG. 38A, line b. It is seen that in the presence of 20 mM Li3PO4,measured Tm values for T30177 increase from 38° C. to 65° C. in therange from 0.1 to 10 mM of added KC1. This very large increase in Tmbelow 10 mM of added KC1, in the presence of 20 mM of Li3PO4 as buffer,argues strongly that the effect of K⁺ binding is not a simple ionicstrength effect.

The inventors have noticed that the measured Tm values for T30177 areconsistently higher, by 1030° C., than has been seen for other smallintramolecular folds (Smith, F. W., & Feigon, J. (1992) Nature (London)344, 410-414; Schultze, et al., J. (1994) J. Mol. Biol. 235, 1532-1547).Since T30177 differs from these other homologues only in terms of theproposed loop domains, the inventors have synthesized homologues ofT30177 where the central G-octet remains constant, but where the loopdomains to either side have been modified by addition or replacement ofa single base. In the context of the simple folding model (FIG. 37A) theT30676 homologue is identical to T30177, but has been modified so as toadd an additional G into the topmost loop of the structure. As seen inFIG. 38A, line c, this one base addition produces a 20° C. decrease inTm over the entire range of K⁺ ion tested. Similarly, the T30677 (SEQ.ID. NO. 55) homologue was prepared (FIG. 37A), which is identical toT30177 (pPT variant of SEQ. ID. NO. 33), but has been modified so as toconvert a pair of Gs in the bottommost loop domain. As seen in FIG. 38A,line d, this two base loop substitution produces a 30° C. decrease in Tmover the range of K⁺ ion tested.

In the context of these substantial stability changes, the inventorssought to confirm that the general mechanism of folding had not beenaltered by base substitution. Thus, Tm analysis was repeated at 1 mM KC1as a function of strand concentration in the range from 3 to 10 μM (FIG.38C). As seen, a measurable strand concentration dependence could not bedetected over this three fold range of variation, for any of thederivatives, thus verifying that the folding equilibrium remainsintramolecular throughout. This was confirmed by native gelelectrophoresis, which continued to display a single folded oligomerstate (not shown), similarly, it was observed that the thermaldifference spectrum for all three homologues was very similar (notshown).

Oligomer Design Improvement

Based upon the unusually high thermal stability of T30177, relative tointramolecular folds in the literature, and upon the 20-40° C. decreasein Tm observed as a function of what should have been a simple loopmodification (FIG. 37A), the inventors concluded that interactionswithin the loop domains may contribute to stability. Specifically, it isproposed that K⁺ ions may engage in stable binding to the loop domainsof T30177. Simple docking calculations, performed with BIOSYM software(not shown) suggested that the TGTG loop configuration at the lower faceof these folded homologues could, in cooperation with the proximalG-tetrad, give rise to tight K⁺ coordination which is similar to thatseen when K⁺ (or Na⁺) ion coordinates between G-tetrads (Bishop et al.,(1996) J. Biol. Chem. 271, 5698-5703). In the context of that proposal,the inventors noticed that, if the penultimate T were removed from the5′ terminus of T30177, the distribution of nucleotide bases in theuppermost face of the fold would be similar to that of the lower face,but with one less internucleotidyl phosphate linkage.

Those considerations served as the basis for the design of the 16 meroligonucleotide, T30695 (FIG. 37A). As seen in FIG. 38A, line a, eventhough T30695 is one base shorter than the T30177 homologue, it wasfound to melt at approximately 10° C. higher temperature, over theentire K⁺ range tested. As for the other homologues, Tm values forT30695 were found to be strand concentration independent, confining thegeneral similarity of the folding process (FIG. 38C).

For T30695, the K⁺ ion dependence of thermal stability was verystriking. In the presence of 20 mM of Li3PO4 as buffer, measured Tmvalues increase from 40° C. to 65° C. over the added KC1 range from 50EM to 1 mM. Again, this ion dependence argues that the observedstabilization is likely to result from site-specific ion binding, ratherthan simple ion-screening effects.

In order to explore the selectivity of ion binding by T30695, Tm valueshave been measured for alkaline metal ions with differing radius: Na⁺(0.99A), K⁺ (1.38A), Rb⁺ (1.49A), and Cs+ (1.69A). As seen in FIG. 38B,significant K⁺ ion selectivity is detected. Although Rb⁺ is very similarto K⁺ in general chemical properties, and differs by only +0.11A in ionradius, it is seen that the Rb⁺ complex with T30695 melts atapproximately 20-30° C. lower temperature over the entire concentrationrange studied. Na⁺ ion and Cs+ ion, which differ from K⁺ in ion radiusby −0.37A and +0.29A, respectively, are seen to be even moredestabilizing. Similar ion binding selectivity were obtained by thismethod for the T30177 homologue (not shown).

Circular Dichroism

In order to explore the nature of these ion binding effects, theinventors monitored the folding of T30695 by circular dichroism (CD)methods. It is known that G-quartet based folding, both intra andintermolecular, gives rise to large induced ellipticity values(Balagurumoorthy, P. & Brahmachari, S. K. (1994) J. Biol. Chem. 269,21858-21869; Jin, et al. (1992) Proc. Natl. Acad. Sci. USA 89,8832-8836; Lu et al. (1992) Biochemistry 31, 2455-2459; Gray et al.(1992) Methods in Enzymology 211, 389-406). Stable tetrad folds arecharacterized by nonconservative spectra, with maxima at 264 nm (˜1×10+5deg-cm2/dmol) and 210 rim (˜5×10+4 deg-cm2/dmol) and a minima at 240 rim(−4×1044 deg-cm2/dmol).

In FIG. 39A, the inventors monitored the CD spectrum of T30695 at 0,0.05 and 10rnM KC1. As seen, at the highest added KC1 concentration, theinduced CD spectrum is very similar to that predicted for an orderlyG-tetrad based fold. Interestingly, the spectrum obtained in the absenceof added KC1 is indicative of significant folding in the absence ofadded K⁺ ion, at 25° C. in the supporting 20 mM Li3PO4 buffer. Tm datafor T30695 in FIG. 38A suggests an extrapolated Tm near to 20° C. in thelimit of very low added K⁺ ion. That extrapolated value is consistentwith the structure evidenced at 0 mM KC1 in FIG. 39A.

Detailed K⁺ titration data of that kind have been presented in FIG. 39B,for T30695, T30177, and T30676. As seen, all three oligomers displayed agenerally similar increase in elliptic as a function of added K⁺ ionconcentration, which is consistent with the hypothesis that they foldire a fashion similar to the simple model of FIG. 37A. However, the ionconcentration dependence of the folding process is quantitativelydifferent for the three. As would have been predicted from the Tm dataof FIG. 38A, it was found that the coupling between K⁺ ion binding andfolding is stronger for T30695 (transition midpoint near to 0.02 mM),than is the case for T30177 (0.15 mM) or T30676 (0.27 mM). The T30677oligomer, which was the least stable of the oligomers tested by Tmanalysis, showed very little ellipticity change over the 0-100 mM KC1range, and therefore has not been presented in FIG. 39B.

Closer inspection of the data in FIGS. 39A and 39B suggested that the CDtitration for T30695 is biphasic, with a first step completed by 0.1rnM, and a second step which is complete in the 1-2 rnM range. In orderto confirm that the K⁺ induced folding process involves two steps, theinventors have performed CD titrations with different alkaline metalions (FIG. 39C). The inventors Rb⁺ induced folding of T30695 isassociated with an overall ellipticity increase which is very similar tothat induced by K⁺. This argues that the Rb⁺ and K⁺ complexes are foldedin a similar fashion. However, as expected from the Tm differences seenin FIG. 38B, it is observed that Rb⁺ ion induced folding isquantitatively different, occurring only at relatively high added ionconcentration (0.5 rnM midpoint as compared to 0.02 rnM for K⁺). Thisconfirms that Rb⁺ is a much poorer effector of the folding process. Avery similar K⁺ vs. Rb⁺ differential was seen for T30177 (not shown),which suggests that the two oligomers display similar overall ionbinding selectivity.

The biphasic character of the T30695 folding process now easily detectedupon addition of Rb⁺ (FIG. 39C). The magnitude of the CD changeassociated with the first and second ion induced steps are similar forboth K⁺ and Rb⁺, confirming that the folding process has not beensignificantly altered in a qualitative fashion by Rb⁺. For comparison,it was observed that folding of T30695 as a function of Na⁺ ion bindingis not biphasic, and is associated with a total ellipticity increasewhich is no larger than that of the first transition seen in thepresence of K⁺ and Rb⁺ ion. One interpretation of this difference isthat Na⁺ ion is capable of driving the first, but not the second step inthe folding process. This proposal will be discussed below.

Folding Kinetics

In order to investigate the two step folding process in more detail, theinventors have measured the kinetics of oligomer folding, for T30695 andT30177 at 25° C. in the standard 20 rnM Li3PO4 buffer. Data wereobtained by manual addition of K⁺ or Rb⁺ ion at time zero, followed bymeasurement of UV absorbance change at 264 nm, in the 0 to 300 secondtime range. In FIG. 40A, K⁺ ion has been added to T30177 at 0.2 uM(curve a), 1.0 rnM (curve b) or 10 uM (curve c). In FIG. 40B, Rb⁺ ionhas been added to T30695 at 1 uM (curve a), 5 uM (curve b) or 10 uM(curve c). These three values are approximately those required to obtainthe midpoint, endpoint and ten times the endpoint of the K⁺ induced(FIG. 39B, curve b) or Rb⁺ induced folding process (FIG. 39C). Althoughnot shown, the kinetic data described below were found to be nucleicacid concentration independent over the range from 3-10 uM in strands,confirming that the folding process is intramolecular.

As seen in FIG. 40A, upon addition of K⁺ to T30177 to 0.2 mM (curve a),a single slow kinetic process is detected with a time constant near 18sec. Interestingly, this component is hyperchromic, indicating a netloss of base stacking interaction during this first step of the foldingprocess. Upon addition of sufficient K⁺ to drive the folding transitionto completion (1 uM, curve b), a second kinetic component is detected(˜=15 sec, t2=1×104 sec). The second component is hypochromic,indicative of a net increase in base stacking, and is very slow. Upon anadditional increase of K⁺ ion to 10 μM (curve c), the first kineticcomponent becomes nearly too fast to be detected in the currentapparatus, while the time constant for the second step has decreased toabout 50 sec. Very similar kinetics, but approximately 20-fold slower,have been obtained upon addition of Rb⁺ ion to T30177 (not shown).

In FIG. 40B, the inventors have performed a similar folding analysis onT30695, but with Rb⁺ instead of K⁺ ion. This was done because, forT30695, the kinetics of K⁺ induced folding were too fast to be detectedin the simple optical apparatus employed. As seen in FIG. 40B, uponaddition of Rb⁺ to 1 mM (curve a), a single slow kinetic process isdetected, similar to that obtained at low K⁺ ion concentration withT30695 (Taul=48 sec). Again, this component is hyperchromic, indicatinga net loss of base stacking interaction. Upon addition of sufficient Rb⁺to drive the T30695 folding transition to completion (5 uM, curve b), asecond kinetic component is detected. Again, the second component ishypochromic, indicative of a net increase in base stacking. Uponadditional increase of Rb⁺ ion concentration to 10 μM (curve c), thefirst kinetic component becomes nearly too fast to be detected, whilethe time constant for the second step has decreased from about 60 sec(curve b) to about 16 sec.

Although these initial kinetic data are not sufficient to solve for rateconstants, the absorbance-detected kinetic data for both T30177 andT30695 are consistent with the equilibrium binding data obtained by CD(FIGS. 39B and C). Both techniques suggest that for K⁺ and Rb⁺, the ioninduced oligomer folding process is aphasic. Kinetic data obtained withNa⁺ ion (not shown), suggest that only the first, hyperchromictransition is obtained at any concentration in the 0-200 mM range. Thatobservation is also generally consistent with Na⁺ titration data (FIG.39C). A structural model is proposed below to rationalize thoseobservations.

A Relationship Between Structure and Function

The inventors' interest in T30177 and its derivatives has arisen becausethis class of oligonucleotide is a potent inhibitor of HIV infection inculture (Rando et al., (1994) J. Biol. Chem. 270, 1754-1760; Ojwang etal. (1994) J. AIDS 7, 560-570; Bishop et al., (1996) J. Biol. Chem. 271,5698-5703; Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39,242-635), and in vitro, has been shown herein to be the most potentinhibitor of HIV-1 integrase to have been identified thus far (see alsoOjwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35). InTable D-1, there is provided a catalog of the melting temperatures ofthe closely related set of derivatives used in this study, as an indexof their stability as an intramolecular tetrad-based fold. Stability hasbeen presented at three different added K⁺ ion concentrations, spanninga range of Tm values which differ by 50° C. This was done to ensure thatstability-activity correlations would not be limited to any particularK⁺ ion concentration.

Three kinds of activity data have been presented. Integrase inhibitionby these oligonucleotides has been monitored for both the 3′ exonucleaseand strand transfer activities of the purified HIV-1 integrase (Ojwanget al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35). Data arepresented in Table D-1 as the IC50, in nM of added oligonucleotide.Antiviral activity has been obtained as described herein and elsewhere(Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35), andis presented as the IC50, in nM, of added oligonucleotide.

Inspection of Table D-1 suggests that, relative to any added K⁺ ionconcentration, there is a correlation between thermal stability of thefolded state and the capacity to inhibit the exonuclease or strandtransfer activity of purified HIV-1 integrase. A qualitative correlationis also obtained when comparing thermal stability with measured anti-HIVactivity in cell culture. A relationship between thermal stability andfunction can only be meaningful for folded structures which are verysimilar. However, given the sequence similarity among these fourhomologues in Table D-1, and the similarity of their ion-induced foldingprocess, the correlations are likely to be meaningful.

Conclusions Regarding the Structure/Function Studies

Data were obtained suggesting that the anti-HIV oligonucleotide drugT30177 and its homologue T30695, fold via intramolecular G-tetradformation, to yield a structure which is stabilized by K⁺ ion binding.It is well known from the literature that alkaline metal ions canstabilize G-tetrad formation (Williamson, J. R. (1994) Annul Rev.Biophys. Biomal. Struct. 27, 703-730). What distinguishes the behaviorof these two oligomers is the unusually high stability of the foldedstate (FIG. 38A), the high selectivity shown for K⁺ ion (FIGS. 38B and39C) and the possibility that K⁺ coordination may be strongly coupled toloop structure within the oligonucleotide fold (FIGS. 38A and 39B).Consistent with the idea that ion binding may occur with G-tetrads andwith loops, the inventors have observed that the folding of T30695 andT30177 appears to occur as a two step process, as detected byequilibrium (FIG. 39) and kinetic methods (FIG. 40).

In order to relate these various observations, the inventors have foundit useful to propose a simple, two step folding model (FIG. 37B). Theysuggest that the first, higher affinity ion binding step occurs bycoordination of metal ion with the central-most pair of G-tetrads,thereby generating a core octet which is similar to that seen in relatedintramolecular folds (Williamson, et al. (1989) Cell 59, 871-880;Panyutin, et al. (1990) Proc. Natl. Acad. Sci. USA 87, 867-870; Smith,F. W., & Feigon, J. (1992) Nature (London) 344, 410-414; Schultze, etal. (1994) J. Mol. Biol. 235, 1532-1547). It is proposed that, byanalogy to those other, better understood G-tetrad based structures,this first ion binding step has rather modest selectivity among thealkaline metal ions (Williamson, J. R. (1994) Annul Rev. Biophys.Biomal. Struct. 27, 703-730). The inventors propose that the second stepin the folding process involves binding of additional ion equivalents tothe loop regions of the structure. It is also proposed that this secondprocess, which occurs at higher added ion concentration (FIG. 39) andwhich is associated with the slow kinetic step of FIG. 40, is coupled toa rearrangement of the loop domains to yield two additional sites formetal ion coordination.

In preliminary modeling studies (not shown), the inventors haveconfirmed that orderly structures of the proposed kind can be obtainedin which carbonyl oxygens from T and G base plains are organized in theloops so as to complement the end of the G-octet, resulting inoctahedral coordination of one K⁺ equivalent at each of the twojunctions between loop and core octet domains. It is proposed that thiscapacity for additional K⁺ ion binding is the origin for the remarkablestability of T30695, the corollary being that other homologues describedin this work are less stable because they have lost one or the other ofthe proposed K⁺ coordination sites. A second corollary of the model isthat the high ion selectivity seen for these oligomer folds is dominatedby the structural requirements for ion binding to the loops, rather thanfrom ion binding within the core octet. Preliminary NMR data (Ding &Hogan, unpublished data) suggests that the additional binding stepinvolves 2 equivalents of K⁺, yielding 3 K⁺ equivalents per oligomerfold, at saturation.

Confirmation of this model awaits detailed structure analysis. However,the data at hand (Table D-1) suggest that formation of the ion-selectiveoligomer fold described herein may be a necessary pre-condition foranti-integrase and the overall anti-HIV activity of these compounds. Assuch, refinement of the present folding model could prove useful as thebasis for pharmaceutical improvement.

TABLE D-1 T_(m) (° C.) T_(m) (° C.) T_(m) (° M) IC₅₀ (nM) IC₅₀ (nM) IC₅₀(nM) Oligomers 5′-Sequence-3′ (1 mM KC1) (10 mM KC1) (180 mM KC1)3′-PROC STR.TRA HIV-1RF T30695 GGGTGGGTGGGTGGGT 67 87* 110* 43 ± 17 24 ±4 70 T30177 GTGGTGGGTGGGTGGGT 53 70*  92*  79 ± 24^(a)  49 ± 5^(a) 75^(a) T30676 GTGGTGGGTGTGGTGGGT 33 46  65 148 ± 26  134 ± 16 1000 T30677 GTGGTTGGTGGGTTGGT  17* 27  40 725 620 >40,000    T_(m) with *were obtained by a calculation according to the linear fitting functionsof T_(m) vs. Log[KC1]. The data with ^(a) were previously reported byOjwang et al. (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39,2426-35.).

Pharmacokinetic Studies

E. Single-dose hemodynamic toxicity and pharmacokinetics of a partialphosphorothioate anti-HIV oligonucleotide (AR177) following intravenousinfusion to cynomolgus monkeys

As part of the pre-clinical assessment of AR177, a toxicity study ofAR177 (T30177) was conducted with the objective of establishing thedose-response relationship between intravenous infusion of AR177 andhemodynamic parameters in cynomolgus monkeys. Intravenous infusion isthe proposed route of administration of AR177 to humans. The presentstudy was conducted using the short term infusion protocol recommendedby the Food and Drug Administration (Black et al., 1994), withmeasurement of central blood pressure, serum chemistry, hematology,coagulation factors, complement factors, and plasma AR177concentrations.

Materials and Methods for Pre-Clinical Toxicology Screens

Materials

AR177 was synthesized at Aronex on a Milligen 8800 oligonucleotidesynthesizer, and made into a stock solution at 25 mg/mL in sterilephosphate-buffered saline. AR177 has a molecular weight of 5793 daltons,and is a fully neutralized sodium salt. The structure of AR177 wascharacterized by phosphorus and proton NMR, sequencing, basecomposition, laser Resorption mass spectrometry, anion exchange HPLC andpolyacrylamide gel electrophoresis. The AR177 was approximately 94% pureaccording to HPLC and electrophoretic analysis. All analyses areconsistent with the proposed structure.

For HPLC analysis of plasma AR 177, tris was obtained from Fisher, NaBrand NaCl were obtained from Sigma, and methanol was purchased from J. T.Baker. The Gen-Pak Fax anion-exchange HPLC column (4.6×100 mm; cat. no.#15490) was purchased from Waters.

Dosing

Twelve experimentally naive cynomolgus monkeys were assigned to fourgroups of three animals each. Prior to dosing, each animal was lightlysedated with a combination of ketamine (10 mg/kg) and diazepam (0.5mg/kg), and a catheter was introduced into the femoral artery forrecording central arterial pressure. Monkeys were given a singleintravenous infusion of 5, 20, or 50 mg AR177/kg or saline over tenminutes through a cephalic vein catheter using a Harvard infusion pump.Arterial blood samples were drawn at −10, +10, +20, +40, +60 and +120minutes relative to the initiation of infusion into EDTA-containingtubes for hematology, complement factors, coagulation assay, serumchemistry, and plasma AR177 determination. At 24 hours post-infusion,blood was drawn via the femoral vein into EDTA-containing tubes forthese same parameters. The concentration of AR177 in dosing solutionswas confirmed post experiment by absorbance at 280 nm on aspectrophotometer. For the determination of AR177 by HPLC, the plasmafraction was obtained by low speed centrifugation of blood, and storedat −20° C. until used. Electrocardiograms (ECGs), central pressure, andheart rate were recorded continuously for 120 minutes following theinitiation of dosing. Table E-1 summarizes the study design. The animalswere observed twice daily for pharmacotoxic signs and general healthbeginning two days before dosing and for seven days following dosing.The monkeys were not necropsied at the end of the study.

Serum chemistry parameters

The following were determined: sodium, potassium, chloride, carbondioxide, total bilirubin, direct bilirubin, indirect bilirubin alkalinephosphatase, lactate dehydrogenase, aspartate aminotransferase, alanineaminotransferase, gamma-glutamyltransferase, calcium, phosphorus,glucose, urea nitrogen, creatinine, uric acid total protein, albumin,globulin, cholesterol and triglycerides. The samples were analyzed atSierra Nevada Laboratories (Reno, Nev.).

Hematology and coagulation parameters

The following were determined: red blood cell count and morphology,total and differential white blood cells, hemoglobin, hematocrit,prothrombin time, fibrinogen, mean cell hemoglobin, mean corpuscularvolume, mean corpuscular hemoglobin concentration, platelet count, andactivated partial thromboplastin time. Hematology parameters weredetermined at Sierra Nevada Laboratories (Reno, Nev.).

Complement factors

The complement split product Bb and total hemolytic complement CH50 weredetermined. The choice of measuring the Bb split product, as opposed toother complement factors, was based on a published study showing theinvolvement of the alternative pathway in complement activation inducedby oligonucleotides (Galbraith et al, 1994). Complement determinationswere performed in the laboratory of Dr. Patricia Giclas at theComplement Laboratory, National Jewish Center for Immunology (Denver,Colo.).

HPLC analysis of AR177 plasma concentrations

AR177 was assayed in the plasma using an anion-exchange HPLC method on aWaters HPLC system with a 626 pump, 996 photodiode array detector, 717autosampler and Millennium system software controlled by an NEC Image466 computer. Buffer A consisted of 0.1 M Tris base, 20% methanol, pH12, and Buffer B consisted of 0.1 M Tris base, 1.0 M NaBr, pH 12. Theanion-exchange column (Gen-Pak Fax column) was equilibrated at 80%buffer A/20% buffer B for 30 minutes before each HPLC run. Fiftymicroliters of 0.2˜filtered, neat plasma were analyzed per run. Theelusion conditions were: a) five-minute isoaatic run at 80% A/20% B.during which the majority of the plasma proteins eluted, b) 25-minutelinear gradient to 30% A/70% B during which AR177 elutes, c) five-minuteSocratic run at 30% A/70% B. d) one-minute linear gradient to 100% B. e)two- minute run at 100% B for column clean-up, and fl two-minute lineargradient to 70% A/30% B for the step in the HPLC clean-up. The high pH(12) of the elusion buffers was necessary to dissociate AR177 fromtissue constituents, which bind AR177 tightly around physiological pH.AR177 is completely stable at pH 12. This method can clearly distinguishbetween the full length AR177 and n-1, n-2, etc. species, which arepotential metabolic products. The ultraviolet detection wavelength was260 nm. The flow rate was 0.5 mL/minute in all steps. Column clean upbetween runs was performed by a 500 pL bolus injection of 0.1 M Trisbase, 2 M NaCl, pH 10.5, followed by: a) ten-minute linear gradient to60% A/40% B. b) one-minute linear gradient to 100% B. c) a three-minuteisocratic run at 100% B and d) one-minute linear gradient to 80% A/20%B.

A standard curve was generated by spiking AR177 into cynomolgus monkeyplasma in order to achieve concentrations of 0.04 to 128 μ/mL. Theplasma standards and unspiked plasma (control) were run on theanion-exchange HPLC column using e above conditions. The WatersMillennium software was used to determine the area under the peak foreach AR177 standard at 260 nm. The HPLC peak area versus AR177concentration was plotted using Cricket Graph III 1.5.1 software. Therewere one to three HPLC replicate runs per AR177 standard. The limit ofquantitation was 25 ng/mL (50 pL injection), whereas the limit ofdetection was 5 ng/mL (50 pL injection). The overall correlationcoefficient of the fitted lines on the standard curve plots was greaterthan 0.999 on two different standard curves used in this study. Thestandard curve was linear over an approximate 3,200-fold range. Thevariability of the replicates was 1-2% at all concentrations. There wasone HPLC run per monkey plasma sample. This method was validated.Further details about the method will be published elsewhere (Wallace etal., submitted).

Pharmacokinetic parameters

The volume of distribution (Vd) was calculated by dividing the totaldose administered by the concentration at the end of the infusion(Rowland and Tozer, 1995). The C_(MAX), (maximum concentration) wastaken from the plasma concentration at the conclusion of the ten-minuteintravenous infusion.

Results/Clinical Observations and Hemodynamic Parameters

Aside from an anticoagulant effect described below, there were noindications of significant toxicity. No clearly treatment-relatedchanges in blood pressure (FIG. 41), heart rate (data not shown) orelectrocardiographic activity (data not shown) were observed, no animalsdied following AR177 infusion. One high-dose (50 mg/kg) monkey exhibiteda rise in arterial pressure during the infusion followed by a decline toapproximately 20-30 mm Hg below the pre-infusion blood pressure. Thesechanges are qualitatively similar to, but less pronounced, than thoseseen in monkeys given total phosphorothioateoligonucleotides (Galbraithet al., 1994). Although suggestive of a treatment effect, thealterations in blood pressure in the subject animal could not be clearlydistinguished from normal fluctuations that occurred in other animals,including one control monkey. The only treatment related clinical signwas emesis during the infusion, which occurred in two of the threeanimals in the 50 mg/kg group and all of the monkeys that received the20 mg/kg dose.

Serum Chemistry

There were no changes in any of the serum chemistry parameters thatcould be attributed to AR177.

Hematology

There were no changes in hematology values attributable to AR 177.Neutrophil counts were increased to a similar extent in all groups,including the saline control group, probably as a result of the stressassociated with the experimental procedure (FIG. 42). The characteristicneutropenia and rebound neutrophilia that has been reported with otheroligonucleotides did not occur in the AR177-treated monkeys, which isconsistent with the relatively small changes in complement Bb splitproduct (FIG. 44) and CH50 (FIG. 45) levels.

Coagulation parameters

The most salient effect of AR177 observed in this study was apronounced, albeit transient, dose-dependent, reversible prolongation ofaPTT in the 20 and 50 mg/kg groups, which reflected inhibition of theintrinsic coagulation pathway. There was at least a four-foldprolongation of aPTT in the 20 and 50 mg/kg dose groups at theconclusion of the infusion of AR177. Determination of the upper aPTTvalue was limited by the range of the assay. (See FIG. 43). This changewas reversible in both dose groups. The aPTT was increased beyond theupper limit of the assay in the 50 mg/kg group for all or most of thetwo-hour monitoring period, but had returned to normal by the followingday. Baseline aPTT values were reestablished by two hours aftertermination of dosing with the 20 mg/kg dose. In the 5 mg/kg group, onlya small and transient rise in aPTT was observed, and there was no changein prothrombin time (PT). Similar changes have been observed with otheroligonucleotides, and are believed to be, at least in part, attributableto direct and reversible binding of the oligonucleotide to thrombin(Henry et al., 1994, Pharmaceutical Res. 11: S353, 1994). PT wasaffected to a much lesser extent than aPTT in the 20 and 50 mg AR177/kggroups (data not shown), indicating little or no effect on the extrinsicpathway.

Complement activation

Plasma levels of the complement split product Bb, a marker foractivation of the alternative pathway, were increased 60-85% overbaseline in the 5 mg/kg group, approximately 2-fold over baseline in the20 mg/kg group, and approximately 2- to 4-fold in the 50 mg/kg group atthe end of infusion. (See FIG. 44). The elevation in Bb persistedthrough the duration of the 2-hour monitoring period, but the values hadreturned to normal by the following day. These increases in Bb weresmall in magnitude. There were also small and transient decreases in theCH50 levels (FIG. 45) in the 20 and 50 mg/kg doses, but there was nodose-CH50 level relationship. In confirmation of this minimal change inCH50, AR177 had no effect on complement CH50 at doses up to 236 μ/mLwhen it was tested in vitro in human or cynomolgus monkey plasma. (Seebelow). Thus, a large increase in complement activation, and resultingcharacteristic neutropenia and rebound neutrophilia, that has beenreported with other oligonucleotides (Galbraith et al., 1994) did notoccur in the AR177-treated monkeys (FIG. 42).

Plasma AR177 concentration

Plasma concentrations of AR177 were maximal at the end of the infusionand declined thereafter with an approximate initial half-life of 20-30minutes (FIG. 46). Another more complete study in cynomolgus monkeys hasshown the terminal half-life to be approximately 24 hours (See below).These half-lives are much longer than that reported by Lee et al.((1995) Pharnaceut. Res. 12:1943-1947) in cynomolgus monkeys for GS-522,a 15 mer oligonucleotide that has a tetrad structure similar to AR177.No metabolites of AR 177 could be observed in the plasma at any timepoint or dose. This contrasts with the results of Lee et al. (1995), whofound significant amounts of shorter species of GS522 in monkey plasmafollowing intravenous infusion. The results with AR177 suggest thatAR177 does not undergo metabolism. There was a direct relationshipbetween the AR177 plasma Cmax and the dose that was administered as aten-minute intravenous infusion to the monkeys (FIG. 45). Plasma Cmaxsof 83.2+/−7.2, 397.8 +/−30.8, and 804.7+/−226.3 μg/mL were achieved forthe 5, 20, or 50 mg/kg doses, respectively, at the end of the infusion(+10 minute time point) (Table E-2; FIG. 47).

The initial volume of distribution (Vd) of the three doses ranged from200-248 mL (mean +s.d.) (Table E-2) at the conclusion of the intravenousinfusion. The mean body weight of the monkeys in the AR177 dose groupswas 3.67 kg. Assuming that plasma volume is 4% of body weight (Daviesand Morris, 1993, Pharmaceutical Res. 10: 1093-1095), the plasma volumewould be 147 mL. Thus, the initial Vd was slightly greater than theplasma volume.

In general, there was a direct relationship between the plasmaconcentration of AR177 and aPTT Atomated Partial Thromboplastin Time forthe 5 (FIG. 48) and 20 (FIG. 49) mg/kg doses. For the 50 mg/kg dose(FIG. 50), the aPTT values were off-scale during the two-hour samplingperiod so it was not possible to determine the relationship between theplasma concentration and aPTT. There was a no effect plasma AR177concentration versus aPTT of approximately 60-100 ug AR177/mL, abovewhich there was prolongation of aPTT. Doubling of aPTT was observed atplasma AR177 concentrations of approximately 100-250 ug AR177/mL.Tripling of aPTT was observed at plasma AR177 concentrations ofapproximately 250-300 fig AR177/mL, after which no correlation waspossible because the aPTT values were beyond the aPTT assay range. Thedisappearance of AR177 from plasma was roughly correlated with thereturn of the aPTT to baseline, which is consistent with direct andreversible binding of the oligonucleotide to one or more clottingfactors. By contrast, there did not appear to be a correlation betweenthe plasma concentration of AR177 and complement split product Bb (datanot shown).

In addition to the in vivo study in cynomolgus monkeys, an in vitrostudy was performed which investigated the effect of AR177 on thecoagulation- cascade and complement activity in cynomolgus monkey andhuman plasma (coagulation) and serum (complement), respectively. AR177caused a two-fold increase in aPTT at a concentration between 30 and 59μg/mL of human plasma, whereas the compound caused a two-fold increasein aPTT at a concentration between 118 and 236 μg/mL of cynomolgusmonkey plasma in vitro. AR177 had no effect on thrombin time in humanplasma, but caused approximately a 2.5-fold increase in thrombin time incynomolgus monkey plasma at 236 μg/mL. AR177 had no effect on eitherfibrinogen or complement CH50 at doses up to 236 ug/mL in human orcynomolgus monkey plasma. AR177 caused a 30% increase in prothrombintime in human plasma and approximately a 15% increase in prothrombintime in cynomolgus monkey plasma at 236 μg/mL.

Discussion

Using an identical dosing regimen to that used in previous experimentsthat resulted in profound hemodynamic effects, the present study showedthat AR177 was very safe. Although limited conclusions can be drawn fromthe present study because only one partial phosphorothioate (AR177) wasexamined, it is possible that the lack of the cardiovascular toxicity isdue to the limited number of phosphorothioate linkages (two) in AR177.It is also speculated that the lack of toxicity could be due to thethree-dimensional (i.e. tetrad) shape of AR177 (Rando et al., 1995, J.Biol. Chem. 270: 1754-1760). In confirmation of the lack of toxicity ofAR177 found in the present study, AR177 does not cause toxicity when itis administered as a bolus intravenous injection to cynomolgus monkeysevery other day at 40 mg/kg for a total of 12 doses (see below).

There was minimal activation of the complement system followingadministration of AR177. Small increases (2-4 fold) in plasma Bb levelsoccurred at plasma AR177 concentrations as high as 750 μ/mL after a doseof 50 mg/kg given as a ten-minute intravenous infusion. Minimal changes(−25%) in the CHX levels occurred at plasma AR177 concentrations as highas 750 μg/mL after a dose of 50 mg/kg given as a ten-minute intravenousinfusion. The complement activation that was seen with AR177 at thesehigh doses did not even result in hypotension. By contrast, Galbraith etal. (1994) have reported that GEM91, a 25-mer phosphorothioateoligonucleotide, caused an 80% decrease in complement CH50, a 700%increase in the level of complement C5a, and death in two out of fourmonkeys following the intravenous infusion of 20 mg/kg over ten minutes.The mechanism by which oligonucleotides activate the complement systemis unknown. However, this phenomenon bears resemblance to a similarphenomenon in human patients during dialysis in which contact betweenblood and dialyser membrane induces complement activation and profoundneutropenia (Heierli et al., 1988, Nephrol. Dial. Transplant 3: 773-783;Jacobs et al., 1989, Nephron 52: 119-124). The present work indicatesthat although AR177 induces some minimal complement activation, this isnot translated into the hemodynamic toxicity that has been seen withother oligonucleotides.

AR177 administration resulted in the dose-dependent inhibition of theintrinsic coagulation pathway, reflected by prolongation of aPTT. Theeffect was maximal at the end of infusion and was reversed in parallelwith clearance of AR177 from plasma. The inhibition of coagulation wassignificant at the highest dose level, but marginal and not consideredclinically significant at the 5 mg/kg dose level. An anticoagulanteffect has been reported to be a class effect of oligonucleotides (Henryet al., 1994). The anticoagulant results with AR177 thus agree withresults that have been seen with other oligonucleotides, although AR177is 40-fold less potent than the thrombin-binding aptamer oligonucleotidethat has been reported by Griffin et al. (1993).

In conclusion, AR177 does not cause mortality, cardiovascular toxicity,or alterations in clinical chemistry in cynomolgus monkeys receivingdoses up to 50 mg/kg as a ten-minute intravenous infusion. However,there was a reversible prolongation of coagulation time at doses of 20and 50 mg/kg. Taken together, the data suggest that AR 177 does not havethe hemodynamic toxicities that are associated with totalphosphorothioate oligonucleotides, and can be administered safely as anintravenous infusion over ten minutes.

TABLE E-1 Monkey Dosing Information Dose Monkey Dose level Dose Conc.volume weight (kg) Group Treatment (mg/kg) (mg/mL) (mL/kg) Male Femalemean ± s.d. 1 Placebo 0 0 4.0 1 2 3.67 ± 0.38 2 AR177 5 1.25 4.0 2 14.12 ± 0.83 3 AR177 20 5.0 4.0 2 1 3.67 ± 0.29 4 AR177 50 4.0 4.0 1 23.23 ± 0.76

Table E-1 - Monkey dosing information

Cynomolgus monkeys were given ten-minute, intravenous infusions of 5, 20or 50 mg AR177/kg at a volume of 4 mL/kg.

TABLE E-2 Plasma AR177, Vd, aPTT and complement Bb values at C_(MAX)Dose AR177 plasma a PTT Bb (mg/kg) (μg/mL) Vd (mL) (seconds) (μg/mL) 583.2 ± 7.2 247.6 ± 21.4  45.9 ± 11.4 0.59 ± 0.07 20 397.8 ± 30.8 184.5 ±14.3 >166.8 ± 23.8 1.48 ± 0.44 50  804.7 ± 226.3 200.7 ± 56.4 >170.0 ±34.6 1.28 ± 0.46

Table E-2 - Plasma AR177 Cm, x, aPTT and complement Bb levels

The AR177 plasma C_(MAX), aPTT, and Bb values are the means ± standarddeviations of data at the +10 minute tune point (end of the infusion).The baseline (10 minutes prior to dosing) aPTT levels were 32.1±4.4,41.6, 6.7 and 33.2±4.8 seconds for the 5, 20, and 50 mg/kg doses,respectively (mean ±s.d.). The baseline (10 minutes prior to dosing) Bblevels were 0.44±0.14, 0.78±0.46 and 0.49±0.21 μ/mL for Me 5, 20, and 50mg/kg doses. Volume of distribution (Vd)=dose/plasma C_(MAX), where thedose is the total mg of AR177.

Serum Chemistry Values Pre-dose 60 min 24 hr Pre-dose 60 min 24 hr GroupAlkaline phosphatase (Units/L) Group Lactate deyhdrogenase (Units/L)Saline 315.0 ± 21.6 293.3 ± 209.3  314.0 ± 234.7 Saline  340.0 ± 190.7337.3 ± 133.1 1138.3 ± 1248.1  5 mg/kg  257.7 ± 141.8 256.7 ± 142.  344.7 ± 195.1  5 mg/kg 228.7 ± 14.8 219.3 ± 11.9  799.0 ± 528.7 20mg/kg  269.3 ± 157.3 245.0 ± 121.5  238.0 ± 136.0 20 mg/kg 266.0 ± 74. 242.7 ± 61.5  583.3 ± 177.4 50 mg/kg 143.0 ± 21.1 140.0 ± 33.8  149.3 ±28.7 50 mg/kg 203.3 ± 7.1  189.3 ± 19.7  409.7 ± 18.6  Asparateaminotransferase (Units/L) Alanine aminotransferase (Units/L) Saline 36.3 ± 16.7 36.7 ± 10.0  193.7 ± 188.9 Saline  49.3 ± 27.5 42.7 ± 23.893.3 ± 55.3  5 mg/kg 35.0 ± 7.5 31.7 ± 7.6   258.0 ± 218.4  5 mg/kg 27.7± 3.5 27.0 ± 4.0  148.3 ± 136.9 20 mg/kg 48.7 ± 6.8 51.0 ± 12.3 119.7 ±22.6 20 mglkg  56.3 ± 34.0 49.3 ± 30.0 97.3 ± 19.9 50 mg/kg  39.7 ± 16.541.7 ± 20.2 128.3 ± 21.2 50 mg/kg 31.3 ± 7.4 30.3 ± 10.3 62.7 ± 7.6 Urea nitrogen (mg/dL) Creatinine (mg/dL) Saline 20.7 ± 1.5 20.7 ± 2.3 23.7 ± 2.5 Saline  0.90 ± 0.45 0.63 ± 0.12 0.90 ± 0.10  5 mg/kg 16.7 ±0.6 17.0 ± 1.0  20.7 ± 3.1  5 mg/kg  0.63 ± 0.06 0.60 ± 0.00 0.97 ± 0.0620 mg/kg 21.7 ± 2.9 21.7 ± 2.1  27.3 ± 0.6 20 mg/kg  0.67 ± 0.05 0.67 ±0.15 0.83 ± 0.15 50 mg/kg 14.7 ± 3.5 15.7 ± 2.5  25.0 ± 6.6 50 mg/kg 0.50 ± 0.10 0.53 ± 0.06 0.67 ± 0.15 Serum chemistry was evaluated atpre-dose, and 1 and 24 hours following initiation of intravenous AR177infusion. Values represent the mean ± s.d. of 3 monkeys. Pre-dose 10 min20 min 40 min 60 min 120 min 24 hr Group White blood cells (10³/mm³)Saline 14.1 ± 40  13.6 ± 3.3  13.9 ± 3.0 14.2 ± 2.7  14.0 ± 2.0 17.8 ±2.2  21.1 ± 2.0   5 mg/kg 9.3 ± 1.0 9.2 ± 2.3  9.5 ± 1.7 9.8 ± 1.2  8.6± 3.0 11.3 ± 2.7  13.2 ± 0.2  20 mg/kg 8.1 ± 1.7 12.3 ± 0.3  12.0 ± 0.611.5 ± 2.2   11.8 ± 02.2 14.9 ± 0.7  14.4 ± 5.5  50 mg/kg 6.7 ± 1.4 11.8± 1.7  10.9 ± 0   11.6 ± 3.1  13.8 ± 1.9 21.3 ± 4.0  13.2 ± 0.8 Lymphocytes (10³/mm³) Saline  8.2 ± 1.5 7.9 ± 1.7  8.4 ± 1.0 8.8 ± 0.2 9.2 ± 0.5 7.7 ± 1.5 11.6 ± 5.3   5 mg/kg  6.2 ± 0.8 5.6 ± 10   6.5 ±0.3 6.8 ± 1.1  5.7 ± 1.6 5.9 ± 0.8 5.5 ± 1.6 20 mg/kg  5.0 ± 1.3 8.5 ±0.3  8.3 ± 0.5 8.8 ± 1.9  8.5 ± 1.5 7.9 ± 1.2 5.3 ± 2.5 50 mg/kg  4.1 ±1.0 7.6 ± 0.7  7.7 ± 1.5 8.0 ± 1.1 10.5 ± 0.4 10.9 ± 1.9  5.3 ± 2.4Monocytes (10³/mm³) Saline  0.54 ± 0.25 0.71 ± 0.17  0.62 ± 0.20 0.58 ±0.20  0.59 ± 0.43 0.98 ± 0.53 1.64 ± 1.06  5 mg/kg  0.37 ± 0.09 0.36 ±0.22  0.35 ± 0.13 0.45 ± 0.06  0.36 ± 0.21 0.52 ± 0.30 1.37 ± 1.00 20mg/kg  0.24 ± 0.05 0.66 ± 0.32  0.59 ± 0.30 0.31 ± 0.15  0.60 ± 0.170.59 ± 0.30 0.99 ± 0.59 50 mg/kg  0.16 ± 0.09 0.94 ± 0.69  0.33 ± 0.150.32 ± 0.18  0.32 ± 0.23 0.75 ± 0.48 0.53 ± 0.35 Platelet (10³mm³)Saline 266.8 ± 41.9 251.3 ± 28.3  258.0 ± 47.8 262.3 ± 51.7  270.0 ±47.6 278.0 ± 46.5  183.7 ± 124.5  5 mg/kg 297.3 ± 34.4 362.3 ± 5.8 369.7 ± 14.6 367.3 ± 52.9  371.7 ± 44.5 292.3 ± 33.0  383.0 20 mg/kg272.3 ± 45.8 172.0 178.0 173.0 ± 48.1  206.0 ± 48.1 258.3 ± 29.0  158.7± 132.4 50 mg/kg 329.3 ± 70.8 UA UA 144.0 UA 278.0 ± 151.3 325.3 ± 105.6Hematology was evaluated at pre-dose, at 10, 20, 40, 60 and 120 minutesand 24 hours following initiation of intravenous AR177 infusion. Valuesrepresent the mean ± s.d. of 2 to 3 monkeys. UA = sample unavailable.Red Blood Cell, Hemoglobin and Hematocrit Values Red blood cells(10⁶/mm³) Saline  5.7 ± 0.4 5.4 ± 0.4  5.3 ± 0.4 5.3 ± 0.4  5.2 ± 0.55.2 ± 0.6 4.7 ± 0.6  5  5.1 ± 0.5 4.9 ± 0.4  5.1 ± 0.6 5.1 ± 0.5  5.1 ±0.5 5.0 ± 0.5 4.7 ± 0.4 20  5.5 ± 0.4 5.1 ± 0.4  5.4 ± 0.2 5.2 ± 0.2 4.9 ± 0.3 5.0 ± 0.3 4.0 ± 0.8 50  5.3 ± 0.4 5.1 ± 0.3  5.4 ± 0.1 4.9 ±0.6  5.1 ± 0.4 4.9 ± 0.8 3.7 ± 1.0 Hemoglobin (g/dL) Saline 13.9 ± 1.613.1 ± 1.3  13.1 ± 1.4 13.2 ± 1.4   128 ± 1.9 12.6 ± 2.0  11.7 ± 1.6   512.3 ± 0.8 11.7 ± 0.5  12.1 ± 0.7 12.2 ± 0.5  12.1 ± 0.7 12.0 ± 0.8 11.3 ± 0.4  20 12.8 ± 0.9 12.3 ± 0.7  12.7 ± 0.9 12.1 ± 0.3  11.6 ± 0.511.9 ± 0.7  9.5 ± 1.7 50 12.7 ± 0.9 12.7 ± 0.7  13.1 ± 0.0 12.1 ± 1.2 12.6 ± 0.8 12.1 ± 1.7  0.2 ± 2.4 Hematocrit (%) Saline 42.1 ± 4.4 40.2 ±2.5  39.9 ± 3.6 40.3 ± 3.6  39.6 ± 4.2 39.0 ± 5.4  35.6 ± 5.1   5 37.8 ±2.0 35.8 ± 1.6  37.1 ± 2.1 37.5 ± 1.3  37.1 ± 20  36.5 ± 1.9  34.1 ±0.4  20 39.6 ± 2.9 37.5 ± 2.2  39.3 ± 2.7 37.4 ± 1.0  35.8 ± 1.3 36.6 ±1.9  29.2 ± 5.4  50 39.8 ± 2.2 393. ± 1.7  40.4 ± 0.4 37.5 ± 3.2  38.9 ±2.3 37.3 ± 5.1  28.1 ± 6.9  Hematology was evaluated at pre-dose, at 10,20, 40, 60 and 120 minutes and 24 hours following initiation ofintravenous AR177 infusion. Values represent the mean ± s.d. of 3monkeys.

F. Repeat-dose toxicity and pharmacokinetics of a partialphosphorothioate anti-HIV oligonucleotide (AR177) following bolusintravenous administration to cynomolgus monkeys

AR177 is a 17-mer partial phosphorothioate oligonucleotide with thesequence 5′GTGGTGGGTGGGTGGGT-3′ (SEQ. ID. NO. 33), with sulfurs at theterminal internucleoside linkages at the 3′ and 5′ ends. It is a potentinhibitor of HIV integrase and HIV production in vitro (Rando et al.,1995; Ojwang et al., 1995), and has a long tissue half-life in rodents(unpublished data). AR177 does not have an antisense- or triplex-basedmechanism of action. A previous study has shown that AR177 does notcause the characteristic hypotension or neutropenia of otheroligonucleotides (Cornish et al., 1993; Galbraith et al., 1994)following a ten-minute intravenous infusion, at doses up to 50 mg/kg(Wallace et al., submitted, 1996). As part of the pre-clinicalassessment of AR177, an intravenous toxicity study of AR177 wasconducted in cynomolgus monkeys with the objective of establishing theclinical and histopathological changes that occur following repeateddoses.

Materials and Methods for Repeat Dose Studies

Materials

For HPLC analysis of plasma AR177 concentrations, tris was obtained fromFisher, NaBr and NaCl were obtained from Sigma, and methanol waspurchased from J. T. Baker. The Gen-Pak Fax anion-exchange HPLC column(4.6×100 mm, cat. no. #15490) was purchased from Waters.

AR177 was synthesized at Biosearch, a division of PerSeptive Biosystems,on a Milligen 8800 oligonucleotide synthesizer, and vialed at 25 mg/mLin phosphate buffered saline. AR177 has a molecular weight of 5793, andis a fully neutralized sodium salt. The structure of AR177 wascharacterized by phosphorus and proton NMR, sequencing, basecomposition, laser Resorption mass spectrometry, anion exchange HPLC andpolyacrylamide gel electrophoresis. All analyses were consistent withthe proposed structure. The AR177 was approximately 94% pure accordingto HPLC and electrophoresis analysis.

The monkeys used in this study were laboratory bred (C.V. Primates,Indonesia or Yunnan National Laboratory, China) and were experimentallynaive prior to the study. The age of the monkeys was 3 to 61/2 years.

Dosing

AR177 was administered intravenously over 1-2 minutes into unsedatedmonkeys every other day for 23 days (12 doses) by injection into thefemoral vein. (See Table E-1). The monkeys were not sedated, but wererestrained during dosing. The highest dose level (40 mg/kg/injection)was selected based on observations in a previous single-dose study ofpronounced anticoagulant activity of AR177 at a dose of 50 mg/kg infusedover 10 minutes (Wallace et al., submitted). A comparable or greaterdegree of anticoagulation was expected to occur with fast (1-2 minute)infusion of 40 mg/kg, and was confirmed by the results of this study.The dosing schedule (every other day) was chosen in order to avoidexcessive accumulation of the test material, which, based onpharmacokinetic data obtained in rats (Wallace et al., submitted), wouldbe expected to occur with daily administration.

The monkeys were observed twice daily for general health, changes inappetite and clinical signs of adverse events. Body weights weremeasured within a few days prior to the first dose (Day 1) andapproximately weekly thereafter. Electrocardiographic (ECG) recordingswere obtained from all animals prior to the study and on Day 22, andfrom recovery animals on Day 35. Blood samples were collected forevaluation of serum chemistry, hematology and coagulation parametersfrom all animals prior to the initiation of the study, on the first dayof dosing (Dose 1; Day 1), and on the last day of dosing (Dose 12; Day23). The sample collection on Days 1 and 23 was timed relative to doseadministration in order to characterize possible acute effects onhematology parameters. An additional clinical pathology evaluation wasconducted for all animals on Day 24, as well as for recovery animals onDay 37. Blood was collected from all animals at 5 minutes, 30 minutesand 4 hours post-dosing on Days 1 and 23 for analysis of the plasmaAR177 concentration.

On Day 25 (two days after the last dose), three males and three femalesfrom each group were humanely euthanized and necropsied, while theremaining two animals each in the high-dose and control groups werecontinued on study for an additional two-week treatment-free “recovery”period, and were euthanized on Day 38. Complete gross necropsies wereperformed on all animals at their scheduled termination. Urine wascollected from each animal during necropsy by bladder puncture andsubmitted for routine urinalysis. Weights of 13 major organs wererecorded, and numerous tissues were collected, preserved and processedfor histology.

Serum chemistry parameters

Serum chemistry was determined pre-study, on day 24 (one day after the12th dose), and on day 37 in the recovery monkeys. The following weredetermined: sodium, potassium, chloride, carbon dioxide, totalbilirubin, direct bilirubin, indirect bilirubin alkaline phosphatase,lactate dehydrogenase, aspartate aminotransferase, alanineaminotransferase, gammaglutamyltransferase, calcium, phosphorus,glucose, urea nitrogen, creatinine, uric acid total protein, albumin,globulin, cholesterol and triglycerides. Serum chemistry was determinedat Sierra Nevada Laboratories (Reno, Nev.).

Hematology and coagulation parameters

Hematology and coagulation parameters were determined 9-11 days prior tothe start of the study, just prior to administering doses 1 (day 1) and12 (day 23), five minutes after dosing (coagulation only), 30 minutesand 4 hours following dosing, one day after the 12th dose (day 24), andin recovery monkeys at sacrifice (day 37). The following weredetermined: red blood cell count and morphology, total and differentialwhite blood cells, hemoglobin, hematocrit, prothrombin time, fibrinogen,mean cell hemoglobin, mean corpuscular volume, mean corpuscularhemoglobin concentration, platelet count, activate partialthromboplastin time, and D-dimer. Hematology was determined at SierraNevada Laboratories (Reno, Nev.).

AR177 plasma HPLC analysis

Blood was taken for plasma analysis of AR 177 just prior to, and at 5,30 and 240 minutes following administration of doses 1 and 12. Theplasma fraction was obtained by low speed centrifugation of blood, andstored at −20° C. until analyzed for the AR177 concentration. PlasmaAR177 concentrations were assayed using an anion-exchange HPLC method ona Waters HPLC system with a 626 pump, 996 photodiode array detector, 717autosampler and Millennium system software controlled by an NEC Image466 computer. Buffer A was 0.1 M Tris base, 20% methanol, pH 12, andBuffer B was 0.1 M Tris base, 1.0 M NaBr, pH 12. anion-exchange columntGen-Pak Fax column) was equilibrated at 80% buffer A/20% buffer B for30 minutes before each HPLC run. Fifty microliters of plasma wereanalyzed per run. The elusion conditions were: a) five-minute isocraticrun at 80% A/20% B. during which the majority of the plasma proteinseluted, b) 30-minute linear gradient to 30% A/70% B during which theAR177 eluted, c) five-minute isocratic run at 30% A/70% B. d) one minutelinear gradient to 100% B. e) two- minute run at 100% B for initialcolumn cleanup, and fl two-minute linear gradient to 70% A/30% B for theinitial step in the clean-up method for HPLC column clean-up. The highpH (12) of the elusion buffers was necessary to dissociate AR177 fromtissue constituents, which bind AR177 tightly around physiological pH.AR177 is completely stable at pH 12. This method can clearly distinguishbetween the full length AR177 and n-1, n-2, etc. species, which arepotential metabolic products. The W detection wavelength was 260 nm. Theflow rate was 0.5 mL/minute in all steps. All runs were performed atroom temperature. Column clean up between runs was performed by a 500 μLbolus injection of 0.1 M Tris base, 2 M NaCl, pH 10.5, followed by: a)ten-minute linear gradient to 60% A/40% B. b) one-minute linear gradientto 100% B. c) a three-minute isocratic run at 100% B and d) one-minutelinear gradient to 80% A/20% B.

AR177 was spiked into cynomolgus monkey plasma in order to achieveconcentrations of 0.0635 to 125 μg/mL for the standard curve. The plasmastandards and unspiked plasma (control) were run on the anion-exchangeHPLC column using the above conditions. The Waters Millennium softwarewas used to determine the area under the peak for each AR177 standard at260 nm. The HPLC area versus AR177 concentration was plotted usingCricket Graph m 1.5.1 software. There were two HPLC replicate runs perAR177 standard. The areas which represented the lowest concentrationwere at least two times the background area at 260 nm. The overallcorrelation coefficient of the fitted lines on the standard curve plotswas greater than 0.999. There was a linear concentration versus A260relationship over a minimum 6,500 fold range. The variability of thereplicates was 1-2%. This method was validated.

Necropsy and histopathology

A complete necropsy was conducted on all monkeys, and includedexamination of the external surface of body (body orifices; dosing site;cranial, nasal, paranasal, thoracic, abdominal and pelvic cavities), andthe external surface of the brain and spinal cord. The organ weights ofthe adrenals, epididymies, liver, pituitary, spleen, thyroids,parathyroids, brain, heart, lungs, prostate, testes, uterus, cervix,kidney, ovaries, seminal vesicles, and thymus were recorded.

A histopathological assessment was made of 46 hematoxylin andeosin-stained tissues by a veterinary pathologist. These includedtissues from the cardiovascular, digestive, respiratory, urogenital,lymphoid/hematopoietic, skin/musculoskeletal and nervous systems, andall major organs.

Results

Clinical observations

No animals died during the course of the study, and there were noeffects on body weight. The only treatment-related clinical sign was anincidence of discoloration around the eyes in three high-dose animals,which occurred on only one occasion (Day 16 or 18) for two of theanimals, and on four consecutive days (Days 18-21) in the third animal.The latter monkey also had swelling around the eyes on Day 18. Thereaction was transient and was limited to the high-dose group.

ECG, clinical chemistry, urinalysis and hematology

No abnormalities in the ECG recordings were noted, and there were notreatment-related changes in serum chemistry or urinalysis parameters.The only changes in hematology parameters considered possiblytreatment-related were an acute and transient increase in lymphocytes inthe high-dose group, and an acute decrease in eosinophils which was seenin all groups, but appeared to be more pronounced in the AR177-treatedgroups. Both of these changes were observed shortly following dosing onDays 1 and 23 (i.e., those days when clinical pathology was evaluated atseveral time points post-dosing), but were largely absent on Day 24 (oneday after the last dose). The values generally remained within thenormal range and were not considered indicative of significant toxicity.

Necropsy and Histopathology

No clearly treatment-related histopathologic changes were seen in anyorgans or tissues, and no effects on organ weight were evident.Eosinophilic material was seen in a few tubules in the medullary area ofthe kidneys of three monkeys in the high dose group on day 25, but wasnot seen in the controls or in the recovery animals. Although this maybe treatment-related, eosinophilic material can sometimes be observed inthe renal tubules of healthy, untreated monkeys.

Plasma AR177 concentration

FIG. 51 shows that there was no difference between the AR177 plasmaconcentrations that were achieved after the first and twelfth (last)doses at either 2.5, 10 or 40 mg/kg. The plasma concentration versustime profile of AR177 is shown in FIG. 52. At the earliest sampling timepoint (five minutes after initiation of dosing), maximal plasma levelsof 35.79±5.99, 135.43±16.19 and 416.54±54.55 μ/mL were achieved for dose#1 at 2.5, 10 and 40 mg/kg (FIG. 52). At the earliest sampling timepoint (five minutes after initiation of dosing), maximal plasma levelsof 33.98+9.98, 113.71±26.55 and 386.39±70.29 μ/mL were achieved for dose#12 at 2.5, 10 and 40 mg/kg. The decay kinetics of the 2.5 mg/kg doseappeared to be different than the decay kinetics of the 10 and 40 mg/kgdoses after either dose 1 (FIG. 52), although no definite conclusionscan be drawn because of the limited number of time points. Nometabolites (i.e. n-1, n-2, etc.) could be observed in the plasma at anytime or any dose. This confirms results in rats showing no metabolism ofAR177 (Wallace et al., submitted).

Coagulation parameters

Dose-dependent anticoagulant activity was manifested at the 10 (FIG. 54)and 40 (FIG. 55) mg/kg doses, whereas there was no anticoagulantactivity following the 2.5 mg/kg dose (FIG. 53). This activity wasevident from the prolongation of activated partial thromboplastin time(aPTT), which reflects a primary effect on the intrinsic coagulationpathway. Following both the 1st and 12th doses, mean aPTT in the 10mg/kg group was increased to approximately twice the pre-dose value by 5minutes post-dosing, but had returned to baseline levels by four hours.Following both the 1st and 12th doses, mean aPTT in the 40 mg/kg groupexceeded the upper limit of the assay five minutes after dosing. By 30minutes post-dosing, aPTT values in the 40 mg/kg group had declined toapproximately 2 to 4-fold above the pre-dose level. By four hours, theaPTT had returned to the pre-dose levels in all but one monkey.

The relationship between the AR177 plasma concentration and aPTT is alsoshown in FIGS. 53, 54, and 55 for doses 2.5, 10, and 40 mg/kg,respectively. There was a no effect plasma AR177 concentration versusaPTT of approximately 60-100 μg AR177/mL, above which there wasprolongation of aPTT. Doubling of aPTT was observed at plasma AR177concentrations of approximately 100-220 μg AR177/mL. Tripling of aPTTwas observed at plasma AR177 concentrations of approximately 220-300 μgAR177/mL, after which no correlation was possible because the aPTTvalues were off-scale. There was no change in aPTT after the first ortwelfth doses of 2.5 mg/kg (FIG. 53), since the AR177 plasmaconcentration did not reach the threshold of approximately 60-100 μg/mL.There was a maximal two-fold increase in aPTT after the first or twelfth10 mg/kg doses (FIG. 54). The elimination kinetics of AR177 and thereturn of aPTT to baseline levels were similar after the first ortwelfth doses.

Discussion

These results indicate that AR177, administered as bolus intravenousinjections up to 40 mg/kg every other day for 12 doses, did not causemortality, histopathological or cardiovascular events that have beendescribed for other oligonucleotides (Galbraith et al., 1994; Srinivasanand Iversen, 1995). The only significant change that was observed was aprolongation of aPTT, which was reversible. To our knowledge, this isthe first oligonucleotide that has not been observed to cause liver andkidney toxicity following intravenous administration.

The structure of AR177 may contribute to its lack of general toxicity.AR177 contains only two phosphorothioate bonds at the 3′ and 5′ termini.These phosphorothioate bonds were designed to help preventendonuclease-induced cleavage of AR177. We speculate that the smallnumber of sulfurs may have reduced the propensity to bind to proteins, aphenomenon that has been observed for full phosphorothioates, which hasbeen speculated to cause toxicity (Srinivasan and Iversen, 1995).AR177's three-dimensional shape may also contribute to its lack oftoxicity. AR177 has been shown to form a structure in which hydrogenbonds form between deoxyguanosine residues to create a “G-tetrad” (Randoet al., 1995). This tetrad structure imparts a compact shape which makesit resistant to degradation (Bishop et al., 1996) and may make itrelatively non-toxic by minimizing reactive sites. The resistance todegradation has been noted in single and repeat dose pharmacokineticsstudies in rodents (Wallace et al., submitted), and in a more completepharmacokinetic study in cynomolgus monkeys which showed a terminalplasma half-life of greater than 24 hours (data not shown).

The results of the AR177 plasma analysis demonstrated that there was nodifference between the AR177 plasma concentrations that were achievedafter the first or twelfth (last) doses of 2.5, 10 or 40 mg/kg. Theseresults can be interpreted to mean that AR177 does not induce metabolicenzymes that would, if they were induced, reduce the concentration ofAR177 by increasing its metabolism. This has the important implicationthat repeat doses of AR177, at least when given every other day for 23days, will not result in pharmacokinetic tolerance.

The results of the AR177 plasma analysis demonstrated that there was aclose relationship between the AR177 plasma concentration and aPTT.There was a no effect plasma AR177 concentration versus aPTT ofapproximately 60-100 μg AR177/mL, above which there was prolongation ofaPTT. The ability to prolong coagulation has been noted to be a featureof other oligonucleotides (Bock et al., 1992; Henry et al., 1994). Anoligonucleotide composed of deoxyguanosines and thymidines has beendescribed that binds to thrombin (Paborsky et al., 1993), demonstratessequence dependent inhibition of coagulation in vitro (Bock et al.1992), has a G-tetrad structure (Wang et al., 1993), and is active as ashort acting anticoagulant in vivo (Griffin et al., 1993; DeAnda et al.,1994). The structure of the oligonucleotide, (GGTTGGTGTGGTTGG) bearssome resemblance to AR177 (GTGGTGGGTGGGTGGGT). Both oligonucleotidesform G-tetrad structures. A comparison of the anticoagulant propertiesof these oligonucleotides indicates that the oligonucleotide isapproximately 10-100 times more potent than AR177. An examination of theanti-HIV properties of the oligonucleotide showed that it had little orno anti-HIV activity (unpublished data). Thus, although botholigonucleotides are composed of deoxyguanosines and thymidine, and formG-tetrads, they have distinct biological properties.

In conclusion, administration of up to 40 mg/kg of AR177 to cynomolgusmonkeys by bolus intravenous injection every other day for 23 days waswell tolerated. No mortality or clinical signs of significant toxicityoccurred. The most salient alteration in clinical pathology parameterswas the prolongation of aPTT in the 10 and 40 mg/kg groups, whichreflects inhibition of the intrinsic coagulation pathway. Theapproximate doubling of aPTT observed in the middle-dose group (10mg/kg) is considered to be marginally clinically significant followingbolus intravenous injection. The severe inhibition of coagulation in the40 mg/kg group may not be dose limiting since aPTT values had returnedto baseline levels fours hours following dosing. It is probable thatprolongation of aPTT at these doses could be circumvented byadministering AR177 as a slow infusion over the course of several hoursin order to stay below the threshold for anticoagulation, which wasestablished to be 60-100 μg/mL of AR177. The absence of clinicalpathology abnormalities or tissue histopathology at even the highestdose (40 mg/kg) after repeated intravenous administration suggests thatthere is little potential for cumulative toxicity with T31077 with anytype of administration.

TABLE F-1 Monkey dosing information Number Dose Dose Dose sacrificed on:level Conc. volume # of animals Day 25 Day 38 Weight (kg) GroupTreatment (mg/kg) (mg/mL) (ML/kg) Male Female (m/f) (m/f) mean ± s.d. 1Placebo 0 0 3.2 3 5 3/3 0/2 3.0 ± 0.6 2 AR177 2.5 0.781 3.2 3 3 3/3 3.0± 0.5 3 AR177 10 3.125 3.2 3 3 3/3 3.2 ± 0.7 4 AR177 40 12.5 3.2 4 4 3/31/1 3.2 ± 0.6 Table 1 - Monkey dosing information. Cynomolgus monkeyswere given bolus intravenous injections of AR177 at 2.5, 10 or 40mg/kg/day at a constant volume every other day for a total of 12 doses.Control monkeys received sterile saline. There were 8 monkeys per group,evenly split between males and females, except for the placebo group,which inadvertently had an extra female. The main group was sacrificedon day 25 # following initiation of dosing, which was two days followingthe twelfth dose on day 23. Two monkeys in the placebo and 40 mg/kggroups were in a recovery group. The recovery group monkeys weresacrificed two weeks (on day 38 after initiation of dosing) after theother monkeys.

TABLE F-2 Body Weights Group Prestudy Day 7 Day 14 Day 21 Day 25 Day 28Day 35 Day 38 Saline 3.0 ± 0.6 3.1 ± 0.6 3.1 ± 0.7 3.1 ± 0.7 3.3 ± 0.62.4 ± 0.2 2.5 ± 0.2 2.5 ± 0.2   2.5 3.1 ± 0.5 3.1 ± 0.6 3.2 ± 0.6 3.2 ±0.6 3.1 ± 0.6 * * * 10 3.2 ± 0.7 3.2 ± 0.7 3.3 ± 0.7 3.3 ± 0.7 3.2 ±0.7 * * * 40 3.2 ± 0.6 3.2 ± 0.6 3.3 ± 0.6 3.2 ± 0.6 3.1 ± 0.6 3.4 ± 0.93.5 * 0.9 3.5 ± 1.0 * All monkeys were sacrificed on day 25. Monkeyswere weighed at pre-study and approximately weekly thereafter. Theweights listed for days 28, 35 and 38 are the recovery group monkeys.Values represent the mean ± s.d. of 2-8 monkeys. There were two monkeysper group in the saline and 40 mg/kg recovery groups, and six monkeysper group in the non-recovery groups.

TABLE F-3 Serum Chemistry Values Prestudy Day 24 Day 37 Prestudy Day 24Day 37 Alkaline phosphatase (Units/L) Lactate dehydrogenase (Units/L)Saline 426.8 ± 194.3 379.4 ± 176.7  222.0 ± 134.4 Saline 506.4 ± 530.61091.6 ± 1057.1 206.0 ± 0.0    2.5 459.0 ± 269.9 389.3 ± 211.5 *   2.5432.5 ± 218.7 625.5 ± 170.2 * 10  19.3 ± 237.1 343.5 ± 184.3 * 10 460.3± 246.7 391.0 ± 151.0 * 40 355.1 ± 194.5 294.6 ± 112.7  289.5 ± 130.8 401985.4 ± 3242.6 764.3 ± 655.7 169.5 ± 20.5 Aspartate aminotransferase(Units/L) Alanine aminotransferase (Units/L) Saline 54.1 ± 62.1 115.3 ±96.8 28.0 ± 7.1 Saline 55.8 ± 23.6 62.6 ± 27.9 58.5 ± 6.4   2.5 59.0 ±56.7 80.5 ± 36.9 *   2.5 54.0 ± 16.5 65.0 ± 15.5 * 10  74.3 ± 798.2 54.0± 15.1 * 10 53.7 ± 13.7 41.2 ± 6.2  * 40 203.3 ± 335.7 160.9 ± 127.324.0 ± 9.9 40  91.3 ± 106.7 61.3 ± 28.5 30.0 ± 57  Blood Urea nitrogen(mg/dL) Creatinine (mg/dL) Saline 16.4 ± 4.5  20.3 ± 5.9  22.5 ± 0.7Saline 0.79 ± 0.14 0.64 ± 0.07  0.50 ± 0.00   2.5 16.2 ± 5.9  22.7 ±5.1  *   2.5 0.78 ± 0.17 0.67 ± 0.10 * 10 19.0 ± 3.2  19.8 ± 2.7  * 100.73 ± 0.05 0.62 ± 0.04 * 40 16.1 ± 3.9  21.4 ± 4.8  17.5 ± 4.9 40 0.79± 0.11 0.71 ± 0.11  0.60 ± 0.14 Serum chemistry was evaluated atpre-study, and at days 24 and 37 (recovery monkeys only) followinginitiation of intravenous AR177 administration. Values represent themean ± s.d. of 2-8 monkeys. There were two monkeys per group in thesaline and 40 mg/kg recovery groups, and six monkeys per group in thenon-recovery groups.

TABLE F-4 White Blood Cell, Lymphocytes, Monocytes and Platelet ValuesDay 1 Day 23 Group Predose 30 min 4 hr Pre-dose 30 min 4 hr Day 24 Day37 White blood cells (10³/mm³) Saline 13.4 ± 2.8 19.4 ± 5.9  17.5 ± 5.1 14.8 ± 5.3  20.6 ± 10.2 18.7 ± 5.3  11.1 ± 3.1  9.8 ± 5.4   2.5 15.7 ±4.3 22.4 ± 4.7  18.5 ± 4.7  13.6 ± 41   18.8 ± 9.1  16.2 ± 6.4  12.4 ±7.2  * 10 11.1 ± 2.7 19.2 ± 4.7  20.1 ± 4.1  11.4 ± 3.2  16.3 ± 4.0 19.1 ± 5.5  9.1 ± 2.5 * 40 16.0 ± 1.7 28.1 ± 4.5  25.0 ± 5.4  12.3 ±2.8  20.1 ± 5.6  20.2 ± 6.0  8.9 ± 2.7 15.4 ± 6.2  Lymphocytes (10³/mm³)Saline  9.4 ± 3.3 7.0 ± 1.8  58 ± 1.7 7.6 ± 3.4 7.0 ± 3.8 5.0 ± 1.0 5.4± 1.7 4.7 ± 1.2   2.5  9.4 ± 3.8 7.0 ± 3.0 6.3 ± 2.4 7.6 ± 2.3 7.2 ± 4.05.1 ± 3.1 5.7 ± 2.5 * 10  7.0 ± 2.4 9.7 ± 2.6 6.9 ± 2.0 6.2 ± 2.5 6.6 ±1.9 4.4 ± 2.0 4.9 ± 1.4 * 40 10.6 ± 2.1 13.8 ± 3.4  6.9 ± 1.4 7.4 ± 2.511.3 ± 4.9  5.8 ± 1.9 4.6 ± 1.5 8.7 ± 5.3 Monocytes (10³/mm³) Saline 0.35 ± 0.25 0.69 ± 0.64 1.00 ± 0.28 0.55 ± 0.38 0.48 ± 0.30 0.89 ± 0.400.44 ± 0.10 0.49 ± 0.31   2.5  0.29 ± 0.26 0.58 ± 0.48 0.88 ± 0.53 0.56± 0.29 0.51 ± 0.47 0.64 ± 0.36 0.45 ± 0.34 * 10  0.36 ± 0.15 0.61 ± 0.60 0.98 ± 0.788 0.43 ± 0.33 0.40 ± 0.20 0.76 ± 0.45 0.39 ± 0.25 * 40  0.37± 0.29 0.92 ± 0.57 1.07 ± 0.45 0.65 ± 0.40 0.72 ± 0.39 1.10 ± 0.74 0.39± 0.24 0.46 ± 0.18 Platelet (10³mm³⁾ Saline 309.0 ± 92.  323.0 ± 100.7296.3 ± 138.2 336.5 ± 81.7  337.3 ± 71.3  334.9 ± 74.6  335.1 ± 87.8 331.5 ± 112.4   2.5 319.5 ± 80.6 321.2 ± 84.8  303.0 ± 77.1  376.5 ±92.0  281.4 ± 110.2 299.6 ± 65.8  341.0 ± 75.6  * 10  391.4 ± 154.3352.2 ± 177.7 337.8 ± 199.5 463.7 ± 137.9 458.3 ± 132.3 417.2 ± 58.5 431.3 ± 85.7  * 40 358.6 ± 52.6 159.5 ± 24.7  337.3 ± 81.6  431.4 ±71.5  353.5 ± 178.9 399.5 ± 65.8  394.4 ± 86.8  393.0 ± 38.2  Leukocytesand platelet hematology were evaluated at predose, 30 minutes and 4hours on days 1 and 23, and on day 24 and 37 following initiation ofintravenous AR177 administration. Values represent the mean ± s.d. of 2to 8 monkeys. (*) = All monkeys were sacrificed on day 25.

TABLE F-5 Red Blood Cell, Reticulocyte, Hemoglobin and Hematocrit ValuesDay 1 Day 23 Group Pre-dose 30 min 4 hr Pre-dose 30 min 4 hr Day 24 Day37 Red blood cells (10³/mm³⁾ Saline 6.0 ± 0.4 5.8 ± 0.4 5.6 ± 0.3 6.0 ±0.6 5.8 ± 0.4 5.6 ± 0.4 4.8 ± 0.4 6.1 ± 0.3   2.5 6.0 ± 0.4 5.6 ± 0.45.5 ± 0.3 6.0 ± 0.5 5.6 ± 0.5 5.4 ± 0.5 4.7 ± 0.4 * 10 5.6 ± 0.5 5.4 ±0.5 5.3 ± 0.4 5.8 ± 0.6 5.6 ± 0.5 5.4 ± 0.4 4.6 ± 0.4 * 40 5.8 ± 0.6 5.5± 0.6 5.4 ± 0.5 5.8 ± 0.7 5.5 ± 0.6 5.3 ± 0.6 4.6 ± 0.6 6.4 ± 0.2Reticulocytes (10⁵/mm³⁾ Saline 0.77 ± 0.29 0.87 ± 0.27 0.82 ± 0.22 1.08± 0.49 0.82 ± 0.37 0.81 ± 0.31 0.72 ± 0.34 0.54 ± 0.23   2.5 0.83 ± 0.210.79 ± 0.26 0.68 ± 0.15 0.97 ± 0.45 0.82 ± 0.24 0.72 ± 0.27 0.84 ±0.32 * 10 0.59 ± 0.16 0.53 ± 0.13 0.66 ± 0.20 1.09 ± 0.31 0.72 ± 0.250.81 ± 0.26 0.63 ± 0.14 * 40 0.99 ± 0.56 0.60 ± 0.20 0.99 ± 0.44 1.09 ±0.33 0.60 ± 0.23 0.87 ± 0.27 0.92 ± 0.33 0.80 ± 0.07 Hemoglobin (g/dL)Saline 13.4 ± 1.4  12.8 ± 1.4  12.3 ± 1.3  13.3 ± 0.7  12.8 ± 1.1  12.4± 1.0  10.8 ± 1.1  11.7 ± 0.2    2.5 14.0 ± 1.1  13.3 ± 1.0  13.0 ± 12. 14.0 ± 0.9  13.3 ± 1.0  12.8 ± 1.0  11.2 ± 0.8  * 10 12.9 ± 0.7  12.5 ±0.9  12.3 ± 0.8  13.2 ± 0.9  12.9 ± 0.8  12.5 ± 1.1  10.6 ± 0.8  * 4013.6 ± 1.4  13.0 ± 1.3  12.7 ± 1.5  13.5 ± 1.2  12.9 ± 1.1  12.3 ± 1.1 10.8 ± 1.4  12.4 ± 2.4  Hematocrit (%) Saline 41.3 ± 3.5  39.6 ± 3.6 37.6 ± 3.3  45.1 ± 1.8  40.1 ± 3.0  38.8 ± 2.2  33.3 ± 3.1  37.5 ± 1.2   2.5 42.6 ± 3.3  40.0 ± 2.4  39.2 ± 2.8  43.1 ± 2.7  40.5 ± 3.1  39.1 ±2.9  34.0 ± 2.4  * 10 38.9 ± 1.9  38.0 ± 2.5  37.2 ± 1.8  40.9 ± 2.6 39.6 ± 2.7  38.1 ± 3.3  32.3 ± 2.0  * 40 41.6 ± 2.8  39.5 ± 3.1  38.6 ±4.0  41.7 ± 3.3  39.8 ± 23.  37.8 ± 3.0  32.7 ± 3.6  39.3 ± 6.2  Redblood cell hematology was evaluated at pre-dose, 30 minutes and 4 hourson days 1 and 23, and on day 24 and 37 following initiation ofintravenous AR177 administration. Values represent the mean ± s.d. of 2to 8 monkeys. (*) = All monkeys were sacrificed on day 25.

G. Human Clinical Trials

Four HIV-infected patients/group were dosed with AR177 at 0.75 mg/kg and115 mg/kg, and two HIV-infected patients were dosed so far with AR177 at3.0 mg/kg by intravenous infusion over two hours.

Methods

Blood was collected in EDTAp coated tubes at 0.25, 0.5, 1, 2, 2.05, 2.5,3, 3.5, 4, 6, 8, 11, 14, 26, 48, 98, and 122 hours following initiationof drug administration. Plasma was obtained by low speed centrifugationof the blood, and was stored frozen until analyzed by HPLC for AR177concentration. The concentration of AR177 was determined in patientplasma using a validated anion-exchange HPLC method at the Division ofClinical Pharmacy of the University of California, San Francisco. Thismethod has a limit of quantitation of 15 ng/mL in human plasma.

Pharmacokinetic analysis

Pharmacokinetic parameters were calculated using PKAnalyst software(MicroMath, Salt Lake City, Utah). The pharmacokinetic data best fit atwo compartment model for all of the patients. The alpha and betahalf-lives were almost identical in each of the patients, based on thesoftware interpretation of the AR177 plasma concentration versus timeplot (FIGS. 56-59). For this reason, only one half-life is reported.(Note that in monkeys, a third half-life of approximately 24 hours wasobserved at a dose of 5 mg/kg given as an intravenous infusion over twohours. A third half-life was not evident in human data, except perhapsfor patient #10.) For each pharmacokinetic parameter, the mean ±s.d. ofn=4 was calculated for the 0.75 and 1.5 mg/kg groups and the mean ±s.d.of n=2 was calculated for the 3.0 mg/kg group.

Results

The plasma concentrations of AR177 following intravenous infusion areshown in FIG. 56 (0.75 mg/kg), FIG. 57 (1.5 mg/kg), FIG. 58 (3.0 mg/kg)and FIG. 59 (all doses). Analysis of this data indicate that the plasmapharmacokinetics of AR177 are not directly proportional to the dose(Table G-1). The increase in the C_(max) and AUC were proportionallymuch greater than the increase in the dose from 0.75 to 3.0 mg/kg. Theincrease in the C_(max) and AUC were much greater than the increase inthe dose. The C_(max) value in the 0.75 mg/kg group was 5.1±1.4 μg/mLand the C_(max) value in the 3.0 mg/kg group was 37.5±0.1 μg/mL,approximately a seven-fold increase (FIG. 60). The AUC value in the 0.75mg/kg group was 703.6±154.7 μg-min/mL and the AUC value in the 3.0 mg/kggroup was 8,277.8±2.937.4 μg-min/mL, approximately a 12-fold increase(Table 1).

The plasma clearance and Vd values reflected the C_(max) and AUC data.The plasma clearance in the 0.75 mg/kg group was 1.1±0.2 mL/min/kg andthe clearance in the 3.0 mg/kg group was 0.4±0.2 mL/min/kg,approximately a 65% decrease (FIG. 61). the initial and steady-statevolumes of distribution in the 0.75 mg/kg group were 0.16±0.05 L/kg and0.14±0.05 L/kg, respectively, whereas the initial and steady-statevolumes of distribution (Vd) in the 3.0 mg/kg group were 0.08±0.00 L/kgand 0.05±0.03 L/kg, respectively (Table 1).

In agreement with the above data, the plasma half-life in the 0.75 mg/kggroup was 28.0±12.7 minutes, and the half-life in the 3.0 mg/kg groupwas 120.1±60.7 minutes, approximately a four fold increase (FIG. 60).

Conclusions

These results indicate that the plasma pharmacokinetics of AR177 arenon-linear and suggest that there is a saturable mechanism for theelimination of the drug.

TABLE G-1 Phase I plasma AR177 pharmacokinetic parameters ParameterPatient #01 Patient #02 Patient #03 Patient #04 mean ± s.d. Dose (mg/kg)0.75 0.75 0.75 0.75 Total dose (mg) 65.1 49.23 50.78 54.23 Body weight(kg) 86.9 65.9 67.1 72.3 C_(MAX) (μg/mL) 5.3 6.7 3.3 5.1 5.1 ± 1.4AUC_(O-infinity)) (μg-min/mL) 730.6 910.2 563.9 609.7 703.6 ± 154.7Terminal T_(½) (min) 24.2 33.3 42.3 12.5 28.0 ± 12.7 CL (mL/min) 89.154.1 90.0 89.0 80.5 ± 17.6 CL (mL/min/kg) 1.0 0.8 1.3 1.2 1.1 ± 0.2Vd_(init) (L) 12.30 7.40 15.20 10.60 11.38 ± 3.26  Vd_(init) (L/kg) 0.140.11 0.23 0.15 0.16 ± 0.05 Vd_(SS) (L) 11.0 6.3 13.9 8.9 10.02 ± 3.19 Vd_(SS) (L/kg) 0.13 0.10 0.21 0.12 0.14 ± 0.05 AUMC_((O-infinity))(μg-min²/mL) 90197.3 106558.0 86825.3 609803.1 86120.9 ± 18892.0Parameter Patient #05 Patient #06 Patient #07 Patient #08 mean ± s.dDose (mg/kg) 1.5 1.5 1.5 1.5 Total dose (mg) 93.75 93.15 110.7 135.6Body weight (kg) 62.5 62.1 74 90.4 C_(MAX) (μg/mL) 11.6 12.9 11.9 15.212.9 ± 1.6  AUC_((O-infinity)) (μg-min/mL) 1,745.6 1,949.0 1,953.02,794.7 2110.6 ± 466.3  Terminal T_(½) (min) 110.1 75.4 29.3 42.3 64.3 ±36.2 CL (mL/min) 53.7 47.8 56.7 48.5 51.7 ± 4.3  CL (mL/min/kg) 0.9 0.80.8 0.5 0.7 ± 0.1 VD_(init) (L) 8.10 7.20 9.30 8.90 8.38 ± 0.93Vd_(init) (L/kg) 0.13 0.12 0.13 0.10 0.12 ± 0.01 Vd_(SS) (L) 5.40 6.268.76 8.11 7.13 ± 1.57 Vd_(SS) (L/kg) 0.09 0.10 0.12 0.09 0.10 ± 0.01AUMC_((O-infinity)) (μg-min²/mL) 175479.1 255258.6 301785.9 467118.0299910.4 ± 123070.2 Parameter Patient #09 Patient #10 Patient #11Patient #12 mean ± s.d Dose (mg/kg) 3 3 Total dose (mg) 224.7 233.4 Bodyweight (kg) 74.9 77.8 C_(MAX) (μg/mL) 37.4 37.6 37.5 ± 0.1 AUC_((O-infinity)) (μg-min/,L) 6,200.8 10,354.9 8277.8 ± 2937.4 TerminalT_(½) (min) 163.0 77.2 120.1 ± 60.7  CL (mL/min) 36.2 22.5 29.4 ± 9.7 CL (mL/min/kg) 0.5 0.3 0.4 ± 0.1 Vd_(init) (L) 6.00 6.20 6.10 ± 0.14Vd_(init) (L/kg) 0.08 0.08 0.08 ± 0.00 Vd_(SS) (L) 1.58 5.40 3.49 ± 2.70Vd_(SS) (L/kg) 0.02 0.07 0.05 ± 0.03 AUMC_((O-infinity)) (μg-min₂/mL)269622.5 2478968.5 1374295.5 ± 1562243.5

Multi-Dose Trials

Zintevir™ (AR177; T30177) was next used in multiple dosing experimentswith AIDS patients. Supporting rationale include:

anti-HIV-1 activity at sub-micromolar concentrations in lymphocytesinfected with clinical isolates of HIV-1;

prevention of cytopathic effects if HIV-1 in primary CD₄+T-celllymphocytes;

activity at high multiplicities of infection (MOI); and

a novel mechanism of action that does not involve inhibition of eitherreverse transcriptase (RT) or protease activity.

Study Design

This was an open-label, single-center, study to evaluate the safety,pharmacokinetic profile and virologic/immunologic activity of AR177 inHIV patients. Patients that met the screening criteria received multipledoses of AR177 infused every other day for 14 days (seven doses).Patients were allowed to participate in the study at ONLY one doselevel. Patients were confined to the Research Unit from Day 0 throughDay 18. Patient activities outside the unit had to be acceptable to, andagreed upon prior to study initiation.

Drugs

AR177 was provided by Aronex Pharmaceuticals, Inc. AR177 was obtainedfrom multiple lots during the course of the study. The study drug wasavailable in two vial sizes. These clear glass vials contained 2.2 cc or15.9 cc of product. Each ml of active drug will deliver a 25 mg dose;thus, the expected total mg dose per vial is 55.0 milligrams and 397.50milligrams, respectively.

Dosages

Patients meeting all entry criteria were given a two-hour continuousinfusion of AR177 every other day for a total of seven infusions. Thedosing schedule utilized is shown in the following table (G-2).

DOSAGE SCHEDULE Group Study Medication Dose Level Number of Patients  1AR177 1.5 mg/kg 3 *2 AR177 3.0 mg/kg 8 *Escalation will occur at a 100%increment from 1.5 mg/kg (Group 1) to 3.0 mg/kg (Group 2), if no ≧GradeIII toxicity(ies) occurs.

Dose escalation occurred at a 100% increment from the starting does of1.50 mg/kg (Group 1) to 3.0 mg/kg (Group 2), if no ≧Grade III toxicitiesare observed. Details regarding does escalation and/or decalation, andthe number of patients to be enrolled at each dose level if toxicity isobserved, was determined.

DOSE ADMINISTRATION

The intravenous infusion of study medication will be administeredcontinuously via an indwelling I.V. catheter at a rate of 2 mL/min fortwo hours.

RESULTS

The plasma levels of HIV-1 RNA are an accepted measure of the plasmaviral titer and are directly related to the progression of HIV infectionto acquired immunodeficiency disease syndrome (AIDS) and death inhumans. Mellors et al. (1996) Science 272:1167-1170. Striking resultswere obtained over the course of a 14-day treatment. In each of thethree patients given 3.0 mg/kg dosages, viral load was significantlyreduced.

TABLE G-3 Viral Load Data Plasma PCR/HIV-1 RNA Number or copies/ml) DoseLevel: 3.0 mg/kg Patient Day 18 I.D. No. Day 0 Day 7 Day 13 % Decrease(4 days post) MMJ/038 111,560 85,020 74,350 33% 106,190 RLV/050  21,40024,380 18,890 12%  26,450 RAH/055 187,740 152-820 84,580 55% 114,260

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned as well as those inherent therein. Theoligonucleotides, compounds, methods, procedures and techniquesdescribed herein are presently representative of preferred embodiments,are intended to be exemplary and are not intended as limitations on thescope. Changes therein and other uses will occur to those skilled in theart which are encompassed within the spirit of the invention or definedby the scope of the appended claims. It will be readily apparent to oneskilled in the art that various substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

87 38 base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 38 /note= “Amine moiety attached to 3′ end” 1 TGGGTGGGGTGGGGTGGGGG GGTGTGGGGT GTGGGGTG 38 38 base pairs nucleic acid singlelinear DNA (genomic) not provided 2 GTGGGGTGTG GGGTGTGGGG GGGTGGGGTGGGGTGGGT 38 18 base pairs nucleic acid single linear DNA (genomic) notprovided 3 GGGTGGGTGG GTGGGTGG 18 38 base pairs nucleic acid singlelinear DNA (genomic) not provided 4 GGTGGTGGGG GGGGGTGGGG TGGTGGTGGGGGTGTTGG 38 36 base pairs nucleic acid single linear DNA (genomic) notprovided 5 GTGGTGGTGG TGTTGGTGGT GGTTTGGGGG GTGGGG 36 36 base pairsnucleic acid single linear DNA (genomic) not provided 6 GTGGTTGGTGGTGGTGTGTG GGTTTGGGGT GGGGGG 36 36 base pairs nucleic acid single linearDNA (genomic) not provided misc_feature 36 /note= “phosphorothioatebackbone” 7 GTGGTGGTGG TGTTGGTGGT GGTTTGGGGG GTGGGG 36 36 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 36/note= “phosphorothioate backbone” 8 GTGGTTGGTG GTGGTGTGTG GGTTTGGGGTGGGGGG 36 31 base pairs nucleic acid single linear DNA (genomic) notprovided 9 GGTGGGGTGG TGGTGGTTGG GGGGGGGGGG T 31 21 base pairs nucleicacid single linear DNA (genomic) not provided 10 GGTGGTTGGG GGGTGGGGGG G21 21 base pairs nucleic acid single linear DNA (genomic) not provided11 GGGTGGGGTG GTGGGTGGGG G 21 30 base pairs nucleic acid single linearDNA (genomic) not provided 12 GGTGGGTGGT TTGTGTGGTT GGTGGGTTTT 30 31base pairs nucleic acid single linear DNA (genomic) not provided 13GGGGGGGGGG TGTGGGGGGG GGTTGTGGTG G 31 27 base pairs nucleic acid singlelinear DNA (genomic) not provided 14 GGTGGGTGGG TTGGGGGGTG GGTGGGG 27 29base pairs nucleic acid single linear DNA (genomic) not provided 15TGGGGTTTGG GTGGGGGGTT GGGTGGTTG 29 24 base pairs nucleic acid singlelinear DNA (genomic) not provided 16 GGGTGGTGGT GTTGGTGTTG TGTG 24 22base pairs nucleic acid single linear DNA (genomic) not provided 17GGTGGGGGGG TTGGTGTGTT TG 22 26 base pairs nucleic acid single linear DNA(genomic) not provided 18 GTGTGGGGGG GTGGGGTGGG GTGGGT 26 26 base pairsnucleic acid single linear DNA (genomic) not provided 19 GGGTGGGTGGGTGGGTGGGT GGGTGG 26 26 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature 26 /note= “Amine moiety attached to3′ end” 20 GTTGGGGGTT GTTGGTGGGG TGGTGG 26 45 base pairs nucleic acidsingle linear DNA (genomic) not provided misc_feature 45 /note= “Aminemoiety attached to 3′ end” 21 TGGTGGGTGT GTGGGGGGTG TTGGGGGTTGTTGGTGGGGT GGTGG 45 45 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature 45 /note= “cholesterol moietyattached to 3′ end” 22 TGGTGGGTGT GTGGGGGGTG TTGGGGGTTG TTGGTGGGGT GGTGG45 45 base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 45 /note= “cholesterol moiety attached to 3′ end” 23GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGTGGG TGGTG 45 45 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 45/note= “Amine moiety attached to 3′ end” 24 GTGGTGGGTG GGTGGGTGGTGGGTGGTGGT TGTGGGTGGG TGGTG 45 26 base pairs nucleic acid single linearDNA (genomic) not provided misc_feature 26 /note= “cholesterol moietyattached to 3′ end” 25 GTTGGGGGTT GTTGGTGGGG TGGTGG 26 45 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 45/note= “Amine moiety attached to 3′ end” 26 GATCCATGTC AGTGACACTGCGTAGATCCG ATGATCCAGT CGATG 45 26 base pairs nucleic acid single linearDNA (genomic) not provided misc_feature 26 /note= “phosphorothioatebackbone and amine moiety attached to backbone” 27 GTTGGGGGTT GTTGGTGGGGTGGTGG 26 26 base pairs nucleic acid single linear DNA (genomic) notprovided 28 GGTGGTGGGG TGGTTGTTGG GGGTTG 26 45 base pairs nucleic acidsingle linear DNA (genomic) not provided 29 GGTGGTGGGG TGGTTGTTGGGGGTTGTTGG GGGTGTGTGG GTGGT 45 18 base pairs nucleic acid single linearDNA (genomic) not provided 30 GGGTGGTTGG GTGGTTGG 18 18 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 18/note= “Amine moiety attached to 3′ end” 31 GGGTGGGTGG GTGGGTGG 18 18base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 18 /note= “Amine moiety attached to 3′ end andphosphothioate backbone” 32 GGGTGGGTGG GTGGGTGG 18 17 base pairs nucleicacid single linear DNA (genomic) not provided misc_feature 17 /note=“Amine moiety attached to 3′ end” 33 GTGGTGGGTG GGTGGGT 17 27 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 27/note= “Amine moiety attached to 3′ end” 34 GTGGTGGGTG GGTGGGTGGTGGGTGGT 27 37 base pairs nucleic acid single linear DNA (genomic) notprovided misc_feature 37 /note= “Amine moiety attached to 3′ end” 35GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGT 37 16 base pairs nucleic acidsingle linear DNA (genomic) not provided misc_feature 16 /note= “Aminemoiety attached to 3′ end” 36 TTGTGGGTGG GTGGTG 16 29 base pairs nucleicacid single linear DNA (genomic) not provided misc_feature 29 /note=“Amine moiety attached to 3′ end” 37 TGGTGGGTGG TGGTTGTGGG TGGGTGGTG 2938 base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 38 /note= “Amine moiety attached to 3′ end” 38 GTGGGTGGGTGGTGGGTGGT GGTTGTGGGT GGGTGGTG 38 45 base pairs nucleic acid singlelinear DNA (genomic) not provided misc_feature 45 /note=“phosphorothioate backbone and amine moiety attached to 3′ end” 39GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGTGGG TGGTG 45 18 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 18/note= “Amine moiety attached to 3′ end” 40 GATCCATGTC AGTGACAC 18 18base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 18 /note= “Amine moiety attached to 3′ end andphosphorothioate backbone” 41 GATCCATGTC AGTGACAC 18 18 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 18/note= “Amine moiety attached to 3′ end” 42 CCCCCCCCCC CCCCCCCC 18 18base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 18 /note= “Amine moiety attached to 3′ end andphosphorothioate backbone” 43 CCCCCCCCCC CCCCCCCC 18 47 base pairsnucleic acid single linear DNA (genomic) not provided 44 TTCATTTGGGAAACCCTTGG AACCTGACTG ACTGGCCGTC GTTTTAC 47 15 base pairs nucleic acidsingle linear DNA (genomic) not provided 45 GTAAAACGAC GGCCA 15 17 basepairs nucleic acid single linear DNA (genomic) not provided 46GTGGTGGGTG GGTGGGG 17 16 base pairs nucleic acid single linear DNA(genomic) not provided 47 GTGGTGGGTG GGTGGG 16 16 base pairs nucleicacid single linear DNA (genomic) not provided 48 TGGTGGGTGG GTGGGT 16 13base pairs nucleic acid single linear DNA (genomic) not provided 49GTGGTGGGTG GGT 13 9 base pairs nucleic acid single linear DNA (genomic)not provided 50 GTGGTGGGT 9 14 base pairs nucleic acid single linear DNA(genomic) not provided 51 GTGGGTGGGT GGGT 14 10 base pairs nucleic acidsingle linear DNA (genomic) not provided 52 GTGGGTGGGT 10 15 base pairsnucleic acid single linear DNA (genomic) not provided 53 GGTTGGTGTGGTTGG 15 17 base pairs nucleic acid single linear DNA (genomic) notprovided 54 GTGGTTGGTG TGGTTGG 17 18 base pairs nucleic acid singlelinear DNA (genomic) not provided 55 GTGGTTGGTG TGGTTGGT 18 18 basepairs nucleic acid single linear DNA (genomic) not provided 56GTGGTGGGTG TGGTTGGT 18 18 base pairs nucleic acid single linear DNA(genomic) not provided 57 GTGGTGGGTG TGGTGGGT 18 17 base pairs nucleicacid single linear DNA (genomic) not provided misc_feature 11 /note=“the base is removed from this nucleotide” 58 GUGGUGGGUG GGUGGGU 17 17base pairs nucleic acid single linear RNA (genomic) not provided 59GUGGUGGGUG GGUGGGU 17 17 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature /note= “the base is removed fromthis nucleotide” 60 GNGGTGGGTG GGTGGGT 17 17 base pairs nucleic acidsingle linear DNA (genomic) not provided 61 GTGGGTGGTG GGTGGGT 17 17base pairs nucleic acid single linear DNA (genomic) not provided 62GTGGTGGGGT GGTGGGT 17 17 base pairs nucleic acid single linear DNA(genomic) not provided 63 GTGGTGGGTGG GGTGGT 17 17 base pairs nucleicacid single linear DNA (genomic) not provided 64 GTGGGTGGTGG GGTGGT 1717 base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature /note= “C-5 propynl dU” 65 GTGGNGGGGT GGTGGGT 17 17 basepairs nucleic acid single linear DNA (genomic) not provided misc_feature13 /note= “C-5 propynl dU” 66 GTGGTGGGTG GGNGGGT 17 17 base pairsnucleic acid single linear DNA (genomic) not provided misc_feature 5,13/note= “C-5 propynl dU” 67 GTGGNGGGTG GGNGGGT 17 17 base pairs nucleicacid single linear DNA (genomic) not provided misc_feature /note= “thebase is removed from this nucleotide” misc_feature 5,13 /note= “C-5propynl dU” 68 GUGGUGGGUG GGUGGGU 17 17 base pairs nucleic acid singlelinear DNA (genomic) not provided misc_feature /note= “the base isremoved from this nucleotide” misc_feature 6,13 /note= “C-5 propynl dU”69 GNGGGTGGTG GGTGGGT 17 17 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature /note= “the base is removed fromthis nucleotide” misc_feature 1,5,6,9,10,13,14,17 /note= “deoxyinosine”70 NNGGNNGGNN GGNNGGN 17 17 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature 6,13 /note= “C-5 propynl dU” 71GNGGGTGGTG GGTGGGT 17 17 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature 13 /note= “3′ cholesterol viatriglycyl linker” 72 GTGGTGGGTG GGTGGGT 17 17 base pairs nucleic acidsingle linear DNA (genomic) not provided misc_feature 13 /note= “5-bromodU” 73 GTGGTGGGTG GGNGGGT 17 17 base pairs nucleic acid single linearDNA (genomic) not provided misc_feature 5,9,13 /note= “5-bromo dU” 74GTGGNGGGNG GGNGGGT 17 17 base pairs nucleic acid single linear DNA(genomic) not provided misc_feature /note= “5-iodo dU” 75 GTGGNGGGTGGGTGGGT 17 17 base pairs nucleic acid single linear DNA (genomic) notprovided misc_feature /note= “5-iodo dU” 76 GTGGTGGGNG GGTGGGT 17 17base pairs nucleic acid single linear DNA (genomic) not providedmisc_feature 13 /note= “5-iodo dU” 77 GTGGTGGGTG GGNGGGT 17 17 basepairs nucleic acid single linear DNA (genomic) not provided misc_feature5,9,13 /note= “5-iodo dU” 78 GTGGNGGGNG GGNGGGT 17 17 base pairs nucleicacid single linear DNA (genomic) not provided 79 GTGGCGGGTG GGTGGGT 1717 base pairs nucleic acid single linear DNA (genomic) not provided 80GTGGTGGGCG GGTGGGT 17 17 base pairs nucleic acid single linear DNA(genomic) not provided 81 GTGGTGGGTG GGCGGGT 17 17 base pairs nucleicacid single linear DNA (genomic) not provided 82 GTGGCGGGCG GGCGGGT 1715 base pairs nucleic acid single linear DNA (genomic) not provided 83TGGGAGGTGG GTCTG 15 15 base pairs nucleic acid single linear DNA(genomic) not provided 84 TGGGAGGTGG GTCTG 15 15 base pairs nucleic acidsingle linear DNA (genomic) not provided 85 TGGGAGGTGG GTCTG 15 20 basepairs nucleic acid single linear DNA (genomic) not provided 86GCGGGGCTCC ATGGGGGTCG 20 17 base pairs nucleic acid single linear DNA(genomic) not provided 87 GTGGTGGGTG GGTGGGT 17

What is claimed is:
 1. An oligonucleotide having a nucleotide sequencechosen from the group consisting of SEQ ID NOS 2-27, 29, 31-39, 46-52and 53-87, wherein said nucleotide sequence is optionally modified atthe 3′ terminus or 5′ terminus by attachment of a substituent moietyselected from the group consisting of propylamine, poly-L-lysine,cholesterol, fatty acid chains of length 2 to 24 carbons, and vitamin E.2. An oligonucleotide having a nucleotide sequence chosen from the groupconsisting of SEQ ID NOS 2-27 29, 31-39, 46-52 and 53-87, wherein saidnucleotide sequence includes at least one modification selected from thegroup consisting of: a modified 3′ terminus by attachment of asubstituent moiety selected from the group consisting of propylamine,poly-L-lysine, cholesterol, fatty acid chains of length 2 to 24 carbons,and vitamin E; a modified 5′ terminus by attachment of a substituentmoiety selected from the group consisting of propylamine, poly-L-lysine,cholesterol, fatty acid chains of length 2 to 24 carbons, and vitamin E;a replacement of at least one phosphodiester moiety with aphosphorothioate moiety; a deletion of a thymidine base from at leastone nucleotide; a substitution of a guanosine for at least onethymidine; a substitution of 5- propynyl-2′-deoxyuridine for at leastone thymidine; a substitution of 5-bromo-2′-deoxyuridine for at leastone thymidine; a substitution of 5-iodo-2′-deoxyuridine for at least onethymidine; a substitution of 2′-deoxyinosine for at least one thymidine;and a substitution of at least one cytidine for at least one thymidine.3. An oligonucleotide having the basic sequence of5′-gtggtgggtgggtgggt-3′  (SEQ ID NO 33, 87), said basic sequenceoptionally modified by addition to or deletion of at least onenucleotide from the 3′ or 5′ terminus such that said oligonucleotide hasa nucleotide sequence from 9 to 45 nucleotides in length; optionallymodified at the 3′ terminus by attachment of a substituent moietyselected from the group consisting of propylamine, poly-L-lysine,cholesterol and cholesterol with a triglycyl linker; optionally modifiedat the 5′ terminus by attachment of a substituent moiety selected fromthe group consisting of propylamine, poly-L-lysine, cholesterol andcholesterol with a triglycyl linker; optionally modified by replacementof at least one phosphodiester moiety with a phosphorothioate moiety;optionally modified by deletion of a thymidine base from at least onenucleotide; optionally modified by substitution of5-1propynyl-2′-deoxyuridine for at least one thymidine, optionallymodified by substitution of5-bromo-2′-deoxyuridine for at least onethymidine, optionally modified by substitution of 5-iodo-2′-deoxyuridinefor at least one thymidine, optionally modified by substitution of2′-deoxyinosine for at least one thymidine, optionally modified bysubstitution of at least one cytidine for at least one thymidine, andoptionally modified by substitution of ribose 2′-O-alkylribose, or2′-arylribose for the deoxyribose of at least one nucleotide.
 4. Theoligonucleotide of claim 3 wherein at least one nucleotide is modifiedby deletion of a thymidine base.
 5. The oligonucleotide of claim 3having the nucleotide sequence and phosphorothioate linkage distribution5′-g*tggtgggtgggtggg*t-3′  (SEQ ID NO 33, 87) wherein * indicates aphosphorothioate linkage.
 6. An oligonucleotide having a sequenceselected from the group consisting of 5′-gtggtgggtgggtgggg-3′ (SEQ ID NO46), 5′-gtggtgggtgggtggg-3′ (SEQ ID NO 47), 5′-ggtgggtgggtgggt-3′ (SEQID NO 48), 5′-gtggtgggtgggt-3′ (SEQ ID NO 49), 5′-gtggtgggt-3′ (SEQ IDNO 50), 5′-   gtgggtgggtgggt-3′ (SEQ ID NO 51),        5′-gtgggtgggt-3′(SEQ ID NO 52), 5′-gtggttggtgtggttgg-3′ (SEQ ID NO 54),5′-ggttggtgtggttggg-3′ (SEQ ID NO 55), 5′-gtggtgggtgtggttggt-3′ (SEQ IDNO 56), 5′-gtggtgggtgtggtgggt-3′ (SEQ ID NO 57), 5′-gtggttggtg_gttggt-3′(SEQ ID NO 55), 5′-gtggtgggtg_ggttggt-3′ (SEQ ID NO 56),5′-gtggttggtg_ggtgggt-3′ (SEQ ID NO 58), 5′-g ggtgggtgggtgggt-3′ (SEQ IDNO 60), 5′-gtgggtggtgggtgggt-3′ (SEQ ID NO 61), 5′-gtggtggggtggtgggt-3′(SEQ ID NO 62), 5′-gtggtgggtggggtggt-3′ (SEQ ID NO 63), and5′-gtgggtggtggggtggt-3′ (SEQ ID NO 64)

wherein_denotes a nucleotide lacking a base; optionally modified at the3′terminus by attachment of a substituent moiety selected from the groupconsisting of propylamine, poly-L-lysine cholesterol and cholesterolwith a triglycyl linker; optionally modified at the 5′terminus byattachment of a substituent moiety selected from the group consisting ofpropylamine poly-L-lysine, cholesterol and cholesterol with a triglycyllinker; optionally modified by replacement of at least onephosphodiester moiety with a phosphorothioate moiety; optionallymodified by deletion of a thymidine base from at least one nucleotide;optionally modified by substitution of 5-propynyl-2′-deoxyuridine for atleast one thymidine, optionally modified by substitution of5-bromo-2′-deoxyuridine for at least one thymidine, optionally modifiedby substitution of 5-iodo -2′-deoxyuridine for at least one thymidine,optionally modified by substitution of 2′-deoxyinosine for at least onethymidine, optionally modified by substitution of at least one cytidinefor at least one thymidine, and optionally modified by substitution ofribose 2′-O-alkylribose, or 2′-O-arylribose for the deoxyribose of atleast one nucleotide.
 7. An oligonucleotide having a sequence selectedfrom the group consisting of 5′-guggugggugggugggu-3′ (SEQ ID NO 59),5′-g_ggtgggtgggtgggt-3′ (SEQ ID NO 60), 5′-g_ggPgggtgggPgggt-3′ (SEQ IDNO 68), 5′-g_gggPggtgggPgggt-3′ (SEQ ID NO 69), 5′-I_ggIIggIIggIIggI-3′(SEQ ID NO 70), 5′-gtgggPggtgggpgggt-3′ (SEQ ID NO 71),5′-gtggtgggtgggtgggt-3′ (SEQ ID NO 72), 5′-gtggtgggtgggBgggt-3′ (SEQ IDNO 73), 5′-gtggBgggBgggBgggt-3′ (SEQ ID NO 74), 5′-gtggIgggtgggtgggt-3′(SEQ ID NO 75), 5′-gtggtgggIgggtgggt-3′ (SEQ ID NO 76),5′-gtggtgggtgggIgggt-3′ (SEQ ID NO 77), 5′-gtggIgggIgggIgggt-3′ (SEQ IDNO 78), 5′-gtggcgggtgggtgggt-3′ (SEQ ID NO 79), 5′-gtggtgggcgggtgggt-3′(SEQ ID NO 80), 5′-gtggtgggtgggcgggt-3′ (SEQ ID NO 81),5′-gtggcgggcgggcgggt-3′ (SEQ ID NO 82), 5′-gggaggtgggtctg-3′ (SEQ ID NO84), 5′-gggaggtgggtctg-3′ (SEQ ID NO 85), 5′-tgggaggtgggtctg-3′ (SEQ IDNO 83), and 5′-gcggggctccatgggggtcg-3′ (SEQ ID NO 86)

wherein P is 5-propynyl-2′-deoxyuridine I is 2′-deoxyinosine B is5-bromo 2′-deoxyuridine, and u is uridine or 2′-O-methyluridine;optionally modified at the 3′terminus by attachment of a substituentmoiety selected from the group consisting of propylamine, poly-L-lysine,cholesterol and cholesterol with a triglycyl linker; optionally modifiedat the 5′terminus by attachment of a substituent moiety selected fromthe group consisting of propylamine, poly-L-lysine cholesterol andcholesterol with a triglycyl linker; and optionally modified byreplacement of at least one phosphodiester moiety with aphosphorothioate moiety.
 8. An oligonucleotide having the nucleotide andphosphorothioate linkage distribution 5′-g*_ggtgggtgggtggg*t-3′  (SEQ IDNO 60) wherein * indicates a phosphorothioate linkage, and _indicates anucleotide lacking a base optionally modified at the 3 ′terminus byattachment of a substituent moiety selected from the group consisting ofpropylamine, poly-L-lysine, cholesterol and cholesterol with a triglycyloptionally modified at the 5′terminus by attachment of a substituentmoiety selected from the group consisting of propylamine, poly-L-lysine,cholesterol and cholesterol with a triglycyl linker; optionally modifiedby replacement of at least one phosphodiester moiety with aphosphorothioate moiety; optionally modified by deletion of a thymidinebase from at least one nucleotide; optionally modified by substitutionof 5-propynyl-2′-deoxyuridine for at least one thymidine, optionallymodified by substitution of 5-bromo-2′-deoxyuridine for at least onethymidine, optionally modified by substitution of 5-iodo-2′-deoxyuridinefor at least one thymidine, optionally modified by substitution of2′-deoxyinosine for at least one thymdine, optionally modified bysubstitution of at least one cytidine for at least one thymidine, andoptionally modified by substitution of ribose 2′-O-alkylribose, or2′-O-arylribose for the deoxyribose of at least one nucleotide.
 9. Anoligonucleotide selected from the group consisting of5′-g*c*ggggc*t*c*c*a*tggggg*t*c*g-3′  (SEQ ID NO 86) and5′-g*cggggctccatgggggtc*g-3′  (SEQ ID NO 86) wherein * indicates aphosphorothioate linkage optionally modified at the 3′terminus byattachment of a substituent moiety selected from the group consisting ofpropylamine, poly-L-lysine, cholesterol and cholesterol with a triglycyllinker; optionally modified at the 5′terminus by attachment of asubstituent moiety selected from the group consisting of propylamine,poly-L-lysine, cholesterol and cholesterol with a triglycyl linker;optionally modified by substitution of 5-propynyl-2′deoxyuridine for atleast one thymidine, optionally modified by substitutionof5-bromo-2′-deoxyuridine for at least one thymidine, optionallymodified by substitution of 5-iodo-2′-deoxyuridine for at least onethymidine, optionally modified by substitution of 2′-deoxyinosine for atleast one thymidine, and optionally modified by substitution of riboseor 2′-O-arylribose for the deoxyribose of at least one nucleotide. 10.An oligonucleotide having the nucleotide sequence5′-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3′  (SEQ ID NO 24),optionally modified by substitution of phosphorothioate forphosphodiester linkages; and modified at the 3′ end by attachment of asubstituent moiety selected from the group consisting of propylamine,poly-L-lysine, cholesterol and similar lipophilic or amine groups whichenhance cellular uptake or stability of the oligonucleotide.
 11. Apharmaceutical composition for inhibiting production of humanimmunodeficiency virus type 1 (HIV-1) comprising an oligonucleotide ofany one of claim 1 and a pharmaceutically acceptable carrier.
 12. Amethod of treating a disease associated with HIV-1 infectionpharmacologically effective dose comprising administering to a person inneed thereof a of the composition of claim 11 said dose being sufficientto inhibit the production of HIV-1 virus.
 13. The method of claim 12wherein said dose is at least 3.0 mg/kg of patient body weight.
 14. Amethod of treating an infection by a human immunodeficiency virus,comprising administering to a person in need thereof a pharmacologicaldose of the composition of claim
 11. 15. The method of claim 14 whereinsaid dose is at least 3.0 mg/kg of patient body weight administered inseven equal doses over 14 days.
 16. The method of claim 14 furthercomprising administering another drug, having a different antiviralmechanism of action than that of said oligonucleotide, optionally beforeor after said administering of said composition such that resistance tosaid other drug or to said composition is avoided.
 17. A method ofmaking a pharmaceutical composition for the treatment of HIV-1 infectioncomprising synthesizing at least one of the oligonucleotides of claim 5or 8, in the form of a fully neutralized salt, dissolving saidoligonucleotide in phosphate-buffered saline; sterilizing saidphosphate-buffered saline solution of said oligonucleotide; andpreparing said solution at a concentration of about 25 mg/mL.